Gram-negative alkaliphilic microorganisms

ABSTRACT

Gram-negative bacteria, which are obligate alkaliphiles, have been isolated from samples of soil, water and sediment and a number of other sources obtained from in and around soda lakes. These bacteria have been analyzed according to the principles of numerical taxonomy with respect to each other, as well as to a variety of known bacteria. In addition, these bacteria are further circumscribed by an analysis of various chemotaxonomic characteristics. The bacteria produce various alkali-tolerant enzymes which may be used in various industrial processes requiring such enzymatic activity in a high pH environment.

This application is a continuation-in-part of U.S. Ser. No. 07/903,786,filed Jun. 24, 1992, now U.S. Pat. No. 5,401,657 which is acontinuation-in-part of U.S. Ser. No. 07/719,307, filed Jun. 24, 1991,abandoned, which is a continuation-in-part of U.S. Ser. No. 07/562,863,filed Aug. 6, 1990 now abandoned all of which are incorporated herein byreference.

The present invention is in the field of microbiology and moreparticularly in the field of alkaliphilic microorganisms.

BACKGROUND OF THE INVENTION

Alkaliphiles are defined as organisms which exhibit optimum growth in analkaline pH environment, particularly in excess of pH 8, and generallyin the range between pH 9 and 10. Alkaliphiles may also be found livingin environments having a pH as high as 12. Obligate alkaliphiles areincapable of growth at neutral pH.

Alkaliphiles may be found in such everyday environments as garden soil,presumably due to transient alkaline conditions caused by biologicalactivity such as ammonification, sulphate reduction or photosynthesis. Amuch richer source of a greater variety of alkaliphilic organisms may befound in naturally occurring, stable alkaline environments such as sodalakes.

A more detailed study of soda lakes and alkaliphilic organisms ingeneral is provided in Grant, W. D., Mwatha, W. E. and Jones, B. E.((1990) FEMS Microbiology Reviews, 75, 255-270), the text of which ishereby incorporated by reference. Lists of alkaline soda lakes may befound in the publications of Grant, W. D. and Tindall, B. J. in Microbesin Extreme Environments, (eds. R. A. Herbert and G. A. Codd); AcademicPress, London, (1986), pp. 22-54; and Tindall, B. J. in HalophilicBacteria, Volume 1, (ed. F. Rodriguez-Valera); CRC Press Inc., BocaRaton, Fla., (1988), pp. 31-70, both texts are also hereby incorporatedby reference.

Alkaliphiles, the majority of which are Bacillus species, have beenisolated from non-saline environments and are discussed by Horikoshi, K.and Akiba, T. in Alkalophilic Microorganisms (Springer-Verlag, Berlin,Heidelberg, N.Y., (1982)). However, alkaliphilic organisms from salineand alkaline environments such as lakes are not discussed therein.Strictly anaerobic bacteria from alkaline, hypersaline, environmentshave been recently described by Shiba, H. in Superbugs (eds. K.Horikoshi and W. D. Grant); Japan Scientific Societies Press, Tokyo andSpringer-Verlag, Berlin, Heidelberg, N.Y., (1991), pp. 191-211; and byNakatsugawa. N., ibid, pp. 212-220.

Soda lakes, which may be found in various locations around the world,are caused by a combination of geological, geographical and climaticconditions. They are characterized by the presence of large amounts ofsodium carbonate (or complexes thereof) formed by evaporativeconcentration, as well as by the corresponding lack of Ca²⁺ and Mg²⁺which would remove carbonate ions as insoluble salts. Other salts suchas NaCl may also concentrate resulting in environments which are bothalkaline and saline.

Despite this apparently harsh environment, soda lakes are neverthelesshome to a large population of prokaryotes, a few types of which maydominate as permanent or seasonal blooms. The organisms range fromalkaliphilic cyanobacteria to haloalkaliphilic archaeobacteria.Moreover, it is not unusual to find common types of alkaliphilicorganisms inhabiting soda lakes in various widely dispersed locationsthroughout the world such as in the East African Rift Valley, in thewestern U.S., Tibet, China and Hungary. For example, natronobacteriahave been isolated and identified in soda lakes located in China (Wang,D. and Tang, Q., "Natronobacterium from Soda Lakes of China" in RecentAdvances in Microbial Ecology (Proceedings of the 5th InternationalSymposium on Microbial Ecology, eds. T. Hattori et al.); JapanScientific Societies Press, Tokyo, (1989), pp. 68-72) and in the westernU.S. (Morth, S. and Tindall, B. J. (1985) System. Appl. Microbiol., 6,247-250). Natronobacteria have also been found in soda lakes located inTibet (W. D. Grant, unpublished observations) and India (Upasani, V. andDesai, S. (1990) Arch. Microbiol., 154, pp. 589-593).

Alkaliphiles have already made an impact in the application ofbiotechnology for the manufacture of consumer products. Alkali-tolerantenzymes produced by alkaliphilic microorganisms have already found usein industrial processes and have considerable economic potential. Forexample, these enzymes are currently used in detergent compositions andin leather tanning, and are foreseen to find applications in the food,waste treatment and textile industries. Additionally, alkaliphiles andtheir enzymes are potentially useful for biotransformations, especiallyin the synthesis of pure enantiomers.

SUMMARY OF THE INVENTION

The present invention provides pure cultures of novel aerobic,Gram-negative alkaliphilic bacteria. These bacteria have been isolatedfrom samples of soil, water, sediment and a number of other sources, allof which were obtained from in and around alkaline soda lakes. Thesealkaliphiles have been analyzed according to the principles of numericaltaxonomy with respect to each other and also to a variety of knownbacteria in order to confirm their novelty. In addition, these bacterialtaxa are further circumscribed by an analysis of various chemotaxonomiccharacteristics.

The present invention also provides data as to the composition of theenvironments from which the samples containing the microorganisms wereobtained, as well as the media required for their efficient isolationand culturing such that one of ordinary skill may easily locate such anenvironment and be able to isolate the organisms of the presentinvention by following the procedures described herein.

It is also an object of the present invention to provide microorganismswhich produce useful alkali-tolerant enzymes, as well as methods forobtaining substantially pure preparations of these enzymes. Theseenzymes are capable of performing their functions at high pH which makesthem uniquely suited for applications requiring such extreme conditions.For example, alkali-tolerant enzymes may be employed in detergentcompositions, in leather tanning and in the food, waste treatment andtextile industries, as well as for biotransformations such as theproduction of pure enantiomers.

The genes encoding these alkali-tolerant enzymes may be isolated, clonedand brought to expression in compatible expression hosts to provide asource of larger volumes of enzyme products which may be, if desired,more easily purified and used in various industrial applications, shouldthe wild-type strain fail to produce sufficient amounts of the desiredenzyme, or does not ferment well.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B. Simplified dendrogram showing clusters (phenons)obtained with the S_(G) coefficient and Unweighted Average Linkageprocedure.

FIGS. 2A and 2B. Simplified dendrogram showing clusters (phenons)obtained with the S_(J) coefficient and Unweighted Average Linkageprocedure.

FIG. 3. Simplified dendrogram obtained with the S_(G) coefficient andUnweighted Average Linkage procedure using the derived minimumdiscriminatory tests.

DETAILED DESCRIPTION OF THE INVENTION Sampling

Several hundreds of strains of bacteria have been isolated from samplesof soil, water, sediment and a number of other sources in and aroundalkaline lakes. These samples were obtained as part of an investigationover a period of three years. The isolated bacteria are non-phototrophiceubacteria. Up until now, such bacteria have not been wellcharacterized.

The samples were collected in sterile plastic bags. Sampling wasconducted at lakes Elmenteita, Nakuru, Bogoria, Crater (Sonachi), LittleNaivasha (Oloidien), Magadi, and Little Magadi (Nasikie Engida), all ofwhich are located in Kenya, East Africa. Alkaline soda lakes havingsimilar environments may also be found in Tibet, China, Hungary and thewestern U.S. At each sampling site, physical parameters such as pH,conductivity and temperature were measured as well as the physicalappearance of the site and the sample. Some of the samples were treatedlocally within 36 hours of collection of the sample but the majoritywere examined offsite, several weeks after collection.

Table 1 lists various strains which have been isolated. The strains arelisted according to the location from which the sample was taken, thephysical appearance of the sample itself and a reference to Table 2which provides the chemical analysis of the lake water samples.

Table 3 provides a list of the isolated strains arranged according tothe results of the numerical taxonomic analysis. Furthermore, Table 3provides physical properties of the sample, in particular thetemperature, conductivity and alkaline pH, as well as the numerousisolation media required for obtaining pure cultures of the newbacteria. These media are letter coded with reference to Appendix A.

Tables 1, 2 and 3 provide data from which the environment of thesampling locations may be characterized. The chemical and physicalanalysis of the samples confirm the presence of alkaline pH, as well asthe presence of unusually high levels of Na₂ CO₃, coupled with lowlevels of Ca²⁺ and Mg²⁺.

No chemical analysis is available for mud samples. Furthermore, nochemical analysis is available for a few samples (see Table 1). However,other samples taken at the same location have been analyzed and aredescribed in Tables 1-3. It is known that the basic environments of sodalakes are stable with respect to their pH and ionic composition.Moreover, the microbial populations found at these sites remain largelystable. Thus, it is to be expected that despite the lack of a chemicalanalysis of certain samples, the environment from which the bacteriawere obtained may nonetheless be determined from the data presented inTables 1-3.

The fresh soda-lake water samples were plated out on an alkalinenutrient medium (Medium A) soon after collection. Microscopic inspectionshowed an unexpectedly high diversity of bacterial types. Consideringthe extremely alkaline nature of the environment, viable counts showedunexpectedly high numbers of organotrophic bacteria, in the range of 10⁵-10⁶ colony forming units per ml. The samples were stored either cooledor at ambient temperatures. After a few weeks' storage, the totalnumbers of bacteria in the sample rose, whereas the diversity of typesdecreased.

                                      TABLE 1                                     __________________________________________________________________________    Alkaliphilic Strains Arranged According to Their Place of origin                                                 ANALYSIS                                   STRAINS  SAMPLE LOCATION                                                                           SAMPLE APPEARANCE                                                                           (Table 2)                                  __________________________________________________________________________    1E.1, 2E.1,                                                                            Lake Elmenteita                                                                           Mud from dried up                                                                           N.R.                                       4E.1, 5E.1,                                                                            (east bay)  lake bed                                                 35E.2, 36E.2,                                                                 37E.2, 38E.2                                                                  wE.5, wEl1,                                                                            Lake Elmenteita                                                                           Sediment and water,                                                                         N.T.                                       wEl2     (east bay)  littoral zone.                                           44E.3, 40E.3,                                                                          Lake Elmenteita                                                                           Mud and water,                                                                              1                                          41E.3, 42E.3,                                                                          (east bay)  littoral zone.                                           44E.3, 53E.4,        Spirulina scum.                                          56E.4                                                                         45E.3, 47E.3,                                                                          Lake Elmenteita                                                                           Brown water   2                                          57E.4    swamp, south-                                                                             and sediment                                                      east arm                                                             48E.3, 58E.4                                                                           Lake Elmenteita                                                                           Grey mud      2                                                   swamp, south-                                                                 east arm                                                             59E.4    Lake Elmenteita,                                                                          Water and sandy                                                                             3                                                   (north-west bay)                                                                          sediment, littoral                                                            zone                                                     16N.1, 17N.1,                                                                          Lake Nakuru,                                                                              Mud and water,                                                                              N.T.                                       18N.1, 19N.1,                                                                          north beach littoral zone.                                           20N.1, 26N.1,                                                                          between Hippo                                                        28N.1, wN1,                                                                            Point and Njoro                                                      wN2, wNk1,                                                                             Point.                                                               wNk2.                                                                         49N.3, 50N.3,                                                                          Lake Nakuru,                                                                              Water column, 4                                          61N.4    north beach littoral zone.                                                    between Hippo                                                                 Point and Njoro                                                               Point.                                                               51N.3, 52N.3                                                                           Lake Nakuru,                                                                              Lake sediment,                                                                              4                                                   north beach littoral zone.                                                    between Hippo                                                                 Point and Njoro                                                               Point.                                                               63N.4    Lake Nakuru;                                                                              Mud and water N.T.                                                water hole, SW                                                                salt flats.                                                          6B.1, 7B.1,                                                                            Lake Bogoria,                                                                             Mud and water,                                                                              N.T.                                       8B.1, 9B.1,                                                                            northern mud                                                                              littoral zone.                                           10B.1, 24B.1,                                                                          flats.                                                               25B.1, wB1,                                                                   wB2, wB4, wB5                                                                 wBn4                                                                          64B.4    Lake Bogoria,                                                                             Dried crust of                                                                              N.R.                                                northern mud                                                                              soda mud.                                                         flats.                                                               65B.4    Lake Bogoria,                                                                             Mud at water  5                                                   northern mud                                                                              line                                                              flats.                                                               wBs4     Lake Bogoria,                                                                             Mud and water,                                                                              N.T.                                                south-west  littoral zone.                                                    shore.                                                               11C.1, 12C.1,                                                                          Crater Lake,                                                                              Mud and water,                                                                              N.T.                                       29C.1,   North point littoral zone.                                           73aC.4, 73bC.4,                                                                        Crater Lake,                                                                              Mud and water,                                                                              6                                          74C.4    North point littoral zone.                                           75C.4    Crater Lake,                                                                              Soda-mud, shore                                                                             N.R.                                                North point line.                                                    77LN.4, 78LN.4                                                                         Little Lake Water column  7                                                   Naivasha,   and sediment                                                      south shore.                                                         21M.1, 22M.1,                                                                          Lake Magadi,                                                                              Mud and water N.T.                                       27M.1    causeway upper                                                                western arm.                                                         92LM.4, 94LM.4                                                                         Little Lake Spring water and                                                                            8                                                   Magadi, north-                                                                            sediment                                                          west springs.                                                        __________________________________________________________________________     N.T. = not tested                                                             N.R. = not relevant                                                      

                                      TABLE 2                                     __________________________________________________________________________    Chemical Analysis of Kenyan Soda Lake Waters                                         Na.sup.+                                                                          K.sup.+                                                                           Ca.sup.2+                                                                         Mg.sup.2+                                                                         SiO.sub.2                                                                         PO.sub.4.sup.3-                                                                   Cl.sup.-                                                                          SO.sub.4.sup.2-                                                                   CO.sub.3.sup.2-                                                                   TON*                               ANALYSIS                                                                             (mM)                                                                              (mM)                                                                              (mM)                                                                              (mM)                                                                              (mM)                                                                              (mM)                                                                              (mM)                                                                              (mM)                                                                              (mM)                                                                              (mM)                                                                              TA§                       __________________________________________________________________________    1      196 3.58                                                                              0.07                                                                              b.l.d.                                                                            2.91                                                                              0.03                                                                              65.1                                                                              2.0 68.0                                                                              0.8 119                            2      140 3.32                                                                              0.48                                                                              0.13                                                                              1.85                                                                              0.02                                                                              46.8                                                                              1.7 32.0                                                                              1.2  86                            3      167 3.32                                                                              0.06                                                                              b.l.d.                                                                            3.10                                                                              0.03                                                                              51.8                                                                              1.7 64.0                                                                              2.2 103                            4      326 5.63                                                                              0.15                                                                              b.l.d.                                                                            3.25                                                                              0.15                                                                              57.5                                                                              0.5 198.3                                                                             1.9 259                            5      735 5.50                                                                              0.21                                                                              0.01                                                                              2.23                                                                              0.09                                                                              100.9                                                                             1.0 476.7                                                                             0.9 612                            6      140 8.95                                                                              0.06                                                                              0.01                                                                              2.13                                                                              0.04                                                                              12.4                                                                              0.8 90.0                                                                              1.1 133                            7      8.7 1.79                                                                              0.28                                                                              0.65                                                                              1.02                                                                              0.003                                                                             4.8 0.5 <10.0                                                                             <0.07                                                                              18                            8      483 4.35                                                                              0.03                                                                              0.03                                                                              0.64                                                                              0.08                                                                              157.8                                                                             1.7 166.0                                                                             1.2 105                            __________________________________________________________________________     b.l.d. = below the limits of detection                                        *TON = Total Organic Nitrogen                                                 §TA = Total Alkalinity in milliequivalents/liter                    

                                      TABLE 3                                     __________________________________________________________________________    Origin of the Strains Arranged by Cluster*                                               SAMPLE                                                                                   Temp.                                                                             Conductivity                                                                         ISOLATION                                    CLUSTER                                                                             STRAIN                                                                             LOCATION                                                                             pH  °C.                                                                        mS/cm  MEDIUM                                       __________________________________________________________________________    1     1E.1.sup.CT                                                                        Elmenteita                                                                           9.5 35  n.t.   A                                            1     2E.1 Elmenteita                                                                           9.5 35  n.t.   A                                            1     wB2  Bogoria                                                                              n.t.                                                                              n.t.                                                                              n.t.   A                                            1     wB5  Bogoria                                                                              n.t.                                                                              n.t.                                                                              n.t.   A                                            1     wBs4 Bogoria                                                                              10.5                                                                              n.t.                                                                              19     A                                            1     10B.1                                                                              Bogoria                                                                              10.5                                                                              36  45     A                                            1     20N.1                                                                              Nakuru 10.5                                                                              36  30-40  A                                            1     27M.1                                                                              Magadi 11.0                                                                              36  100    A                                            1     Comamonas terrigena.sup.T (NCIMB 8193)                                                                   --                                           1     wNk2 Nakuru 10.5                                                                              n.t.                                                                              19     A                                            1     Pseudomonas putida.sup.T (NCIMB 9494)                                                                    --                                           2     39E.3                                                                              Elmenteita                                                                           10-10.5                                                                           23  13.9   M                                            2     41E.3                                                                              Elmenteita                                                                           10-10.5                                                                           23  11.3   N                                            2     45E.3.sup.CT                                                                       Elmenteita                                                                           10  27  11.3   P                                            2     47E.3                                                                              Elmenteita                                                                           10  27  11.3   O                                            2     51N.3                                                                              Nakuru 10-10.5                                                                           29  40.1   P                                            2     52N.3                                                                              Nakuru 10-10.5                                                                           29  40.1   P                                            2     42E.3                                                                              Elmenteita                                                                           10-10.5                                                                           23  13.9   N                                            2     50N.3                                                                              Nakuru 10-10.5                                                                           29  40.1   N                                            2     Pseudomonas stutzeri.sup.T (NCIMB 11358)                                                                 --                                           --    wN2  Nakuru n.t.                                                                              n.t.                                                                              n.t.   A                                            --    Pseudomonas beijerinckii.sup.T (NCIMB 9041)                                                              --                                           --    4E.1 Elmenteita                                                                           9.5 35  n.t.   A                                            --    5E.1 Elmenteita                                                                           9.5 35  n.t.   A                                            3     6B.1 Bogoria                                                                              10.5                                                                              36  45     A                                            3     7B.1 Bogoria                                                                              10.5                                                                              36  45     A                                            3     8B.1 Bogoria                                                                              10.5                                                                              36  45     A                                            3     38E.2                                                                              Elmenteita                                                                           n.t.                                                                              n.t.                                                                              n.t.   B                                            3     56E.4                                                                              Elmenteita                                                                           10-10.5                                                                           23  13.9   C                                            3     25B.1                                                                              Bogoria                                                                              10.5                                                                              36  45     A                                            3     26N.1                                                                              Nakuru 10.5                                                                              36  30-45  A                                            3     11C.1                                                                              Crater 9.0 30  10     A                                            3     wB1  Bogoria                                                                              n.t.                                                                              n.t.                                                                              n.t.   A                                            3     12C.1                                                                              Crater 9.0 30  10     A                                            3     28N.1.sup.CT                                                                       Nakuru 10.5                                                                              36  30-40  A                                            3     61N.4                                                                              Nakuru 10-10.5                                                                           29  40.1   E                                            3     36E.3                                                                              Elmenteita                                                                           n.t.                                                                              n.t.                                                                              n.t.   K                                            3     40E.3                                                                              Elmenteita                                                                           10-10.5                                                                           23  13.9   M                                            3     65B.4                                                                              Bogoria                                                                              n.t.                                                                              n.t.                                                                              41.9   C                                            3     94LM.4                                                                             Little Magadi                                                                        9-9.5                                                                             81  35.0   L                                            3     19N.1                                                                              Nakuru 10.5                                                                              36  30-40  A                                            3     24B.1                                                                              Bogoria                                                                              10.5                                                                              36  45     A                                            3     21M.1                                                                              Magadi 11.0                                                                              36  100    A                                            3     29C.1                                                                              Crater 9.0 30  10     A                                            3     35E.2                                                                              Elmenteita                                                                           n.t.                                                                              n.t.                                                                              n.t.   I                                            3     37E.2                                                                              Elmenteita                                                                           n.t.                                                                              n.t.                                                                              n.t.   J                                            3     48E.3                                                                              Elmenteita                                                                           10.0                                                                              27  11.3   P                                            3     78LN.4                                                                             Little 8.5-9                                                                             30  1.2    G                                                       Naivasha                                                           3     73aC.4                                                                             Crater n.t.                                                                              n.t.                                                                              10.2   D                                            3     75C.4                                                                              Crater n.t.                                                                              n.t.                                                                              n.t.   H                                            3     73bC.4                                                                             Crater n.t.                                                                              n.t.                                                                              10.2   D                                            3     74C.4                                                                              Crater n.t.                                                                              n.t.                                                                              10.2   H                                            3     77LN.4                                                                             Little 8.5-9                                                                             30  1.2    F                                                       Naivasha                                                           3     wN1  Nakuru n.t.                                                                              n.t.                                                                              n.t.   A                                            3     49N.3                                                                              Nakuru 10-10.5                                                                           29  40.1   Q                                            3     44E.3                                                                              Elmenteita                                                                           10.0                                                                              27  13.9   O                                            3     58E.4                                                                              Elmenteita                                                                           10.0                                                                              27  11.3   G                                            3     57E.4                                                                              Elmenteita                                                                           10.0                                                                              27  11.3   C                                            4     wE5  Elmenteita                                                                           n.t.                                                                              n.t.                                                                              n.t.   A                                            4     wB4.sup.CT                                                                         Bogoria                                                                              n.t.                                                                              n.t.                                                                              n.t.   A                                            4     wNk1 Nakuru 10.5                                                                              n.t.                                                                              19     A                                            4     wEl1 Elmenteita                                                                           10.4                                                                              n.t.                                                                              13     A                                            4     wEl2 Elmenteita                                                                           10.4                                                                              n.t.                                                                              13     A                                            5     9B.1 Bogoria                                                                              10.5                                                                              36  45     A                                            5     16N.1                                                                              Nakuru 10.5                                                                              36  30-40  A                                            5     17N.1.sup.CT                                                                       Nakuru 10.5                                                                              36  30-40  A                                            5     22M.1                                                                              Magadi 11.0                                                                              36  100    A                                            6     18N.1                                                                              Nakuru 10.5                                                                              36  30-40  A                                            6     59E.4                                                                              Elmenteita                                                                           10.0                                                                              31-33                                                                             12.7   G                                            6     64B.4.sup.CT                                                                       Bogoria                                                                              n.t.                                                                              n.t.                                                                              n.t.   E                                            6     63N.4                                                                              Nakuru 9.0 n.t.                                                                              n.t.   C                                            6     53E.4                                                                              Elmenteita                                                                           10-10.5                                                                           23  13.9   G                                            --    92LM.4                                                                             Little Magadi                                                                        9-9.5                                                                             81  35.0   L                                            --    wBn5 Bogoria                                                                              10.5                                                                              n.t.                                                                              19     A                                            __________________________________________________________________________     *Clusters of microorganisms are obtained by analysis according to the         principles of numerical taxonomy using the S.sub.G /UPGMA method (see         discussion below and FIG. 1).                                                 n.t. = not tested                                                        

The letter codes given for the Isolation Media refer to Appendix A.

Treatment of the Samples: Enrichment and Isolation of AlkaliphilicBacteria

A wide diversity of enrichment and isolation methods were applied. Someof the methods were specifically designed for the enrichment andisolation of alkaliphilic bacteria which exhibit specific types ofenzyme activity at an alkaline pH. Other techniques of a more generalnature were applied for the isolation of diverse sorts of alkaliphilicbacteria. In some cases, the specific conditions prevailing in the lakes(Table 2) were taken into account when experiments were performed forthe isolation of bacteria.

The different nutrient media employed for the isolation of the newalkaliphilic bacteria are designated Medium A-Medium Q. The compositionof the various media employed is shown in Appendix A.

For the isolation of non-specific alkaliphilic organotrophic bacteria,soda-lake water samples or dilutions thereof were streaked out on analkaline nutrient agar, pH 10-pH 10.5 (Medium A). Samples of a moresolid consistency, mud, sediment, etc. were first suspended in analkaline nutrient broth (Medium A) before spreading on an alkalinenutrient agar (Medium A). The bacteria were cultivated in a heatedincubator, preferably at 37° C. In some cases, the samples weresuspended in an alkaline nutrient broth (Medium A) and the bacteriacultivated by shaking, preferably at 37° C. for 2-3 days beforespreading the broth onto an alkaline nutrient agar (Medium A) for theisolation of bacterial colonies.

For the isolation of alkaliphilic bacteria exhibiting specific types ofenzyme activity, samples were spread onto alkaline nutrient agarcontaining specific substrates such as lactalbumin or casein or oliveoil. In some instances, the bacteria in the sample may be enriched for 1day or several weeks in a non-specific alkaline nutrient broth such asMedium A before spreading the broth onto an alkaline nutrient agarspecific for the detection of bacteria exhibiting enzyme activity suchas lipolytic or proteolytic activity.

Taxonomic Analysis

Seventy strains of bacteria isolated from in and around alkaline lakeswere assigned to the type of bacteria known as Gram-negative bacteria onthe basis of (1) the Dussault modification of the Gram's stainingreaction (Dussault, H. P., (1955), J. Bacteriol., 70, 484-485); (2) theKOH sensitivity test (Gregersen, T., (1978), Eur. J. Appl. Microbiol.and Biotech. 5, 123-127; Halebian, S. et al., (1981), J. Clin.Microbiol., 13, 444-448); (3) the aminopeptidase reaction (Cerny, G.,(1976), Eur. J. Appl. Microbiol., 3, 223-225; ibid, (1978), 5, 113-122);and in many cases, confirmation also on the basis of (4) a quinoneanalysis (Collins, M. D. & Jones, D., (1981), Microbiol. Rev., 45,316-354) using the method described by Collins, M. D. in ChemicalMethods in Bacterial Systematics (eds. M. Goodfellow & D. Minnikin) pp.267-288, Academic Press, London, 1985.

The seventy strains were tested for 104 characters. The results wereanalyzed using the principles of numerical taxonomy (Sneath, P. H. A.and Sokal, R. R., in Numerical Taxonomy, W. H. Freeman & Co., SanFrancisco, 1973). The characters tested and how they were tested arecompiled in Appendix B. In addition, Appendix C records how eachcharacter was coded for taxonomic analysis.

Since there are no well-documented strict or obligate non-phototrophic,alkaliphilic Gram-negative eubacteria known to the inventors, a diversecollection of 20 known Gram-negative bacteria were subjected as controlsto the same analysis, using modified pH conditions. These 20 knownreference bacteria are recorded in Table 4 from which it will be seenthat in most cases the "Type Strain" of the known species has been used.

                  TABLE 4                                                         ______________________________________                                        Gram-Negative Non-Alkaliphilic Reference Strains                              ______________________________________                                        (C.t.)   Comamonas terrigena.sup.T NCIMB 8193                                 (P.p.)   Pseudomonas putida.sup.T NCIMB 9494                                  (P.s.)   Pseudomonas stutzeri.sup.T NCIMB 11358                               (A.t.)   "Alcaligenes tolerans" Leicester University strain                   (V.c.)   Vibrio costicola.sup.T NCIMB 701                                     (P.a.)   Providencia alcalifaciens.sup.T NCTC 10286                           (P.v.)   Proteus vulgaris.sup.NT ATCC 13315                                   (M.v.)   Moellerella wisconsensis.sup.T NCTC 12132                            (E.t.)   Edwardsiella tarda.sup.T NCTC 10396                                  (A.h.)   Aeromonas hydrophila.sup.T NCTC 8049                                 (A.s.)   Aeromonas sp S5 Leicester University strain                          (F.a.)   Flavobacterium aquatile.sup.T NCIMB 8694                             (E.c.)   Escherichia coli.sup.T NCTC 9001                                     (E.a.)   Enterobacter aerogenes.sup.T NCTC 10006                              (K.a.)   Klebsiella pneumonia ATCC 15380                                               ("K. aerogenes")                                                     (H.a.)   Hafnia alvei.sup.T ATCC 13337                                        (C.f.)   Citrobacter freundii.sup.T NCTC 9750                                 (S.m.)   Serratia marcescens.sup.T NCTC 10211                                 (P.b.)   Pseudomonas beijerinckii.sup.T NCIMB 9041                            (H.e.)   Halomonas elongata.sup.T ATCC 33173                                  ______________________________________                                         *abbreviation used in FIGS. 1A and 1B and FIGS. 2A and 2B                     .sup.T denotes "Type Strain"-                                                 .sup.NT denotes "Neotype Strain"-                                        

ANALYSIS OF TEST DATA The Estimation of Taxonomic Resemblance

The phenetic data, consisting of 104 unit characters were scored asindicated in Appendix C, and set out in the form of an "n×t" matrix,whose t columns represent the t bacterial strains to be grouped on thebasis of resemblances, and whose n rows are the unit characters.Taxonomic resemblance of the bacterial strains was estimated by means ofa similarity coefficient (Sneath, P. H. A. and Sokal, R. R., NumericalTaxonomy, supra, pp. 114-187). Although many different coefficients havebeen used for biological classification, only a few have found regularuse in bacteriology. We have chosen to apply two associationcoefficients (Sneath, P. H. A. and Sokal, R. R., ibid, p. 129 et seq.),namely, the Gower and Jaccard coefficients. These have been frequentlyapplied to the analysis of bacteriological data and have a wideacceptance by those skilled in the art since they have been shown toresult in robust classifications.

The coded data were analyzed using the TAXPAK program package (Sackin,M. J., "Programmes for classification and identification". In Methods inMicrobiology, volume 17 (eds. R. R. Colwell and R. Grigorova), pp.459-494, Academic Press, London, (1987)) run on a DEC VAX computer atthe University of Leicester, U.K.

A similarity matrix was constructed for all pairs of strains using theGower Coefficient (S_(G)) with the option of permitting negative matches(Sneath, P. H. A. and Sokal, R. R., supra, pp. 135-136) using theRTBNSIM program in TAXPAK. As the primary instrument of analysis and theone upon which most of the arguments presented herein are based, theGower Coefficient was chosen over other coefficients for generatingsimilarity matrices because it is applicable to all types of charactersor data, namely, two-state, multistate (ordered and qualitative), andquantitative.

Cluster analysis of the similarity matrix was accomplished using theUnweighted Pair Group Method with Arithmetic Averages (UPGMA) algorithm,also known as the Unweighted Average Linkage procedure, by running theSMATCLST sub-routine in TAXPAK.

The result of the cluster analysis is a dendrogram, a simplified versionof which is provided in FIG. 1. The dendrogram illustrates the levels ofsimilarity between the bacterial strains. The dendrogram is obtained byusing the DENDGR program in TAXPAK.

The phenetic data, omitting multistate characters (characters 1-5, 11and 12; Appendix C) and thus consisting of 193 unit characters, andscored in binary notation (positive=1, negative=0) were re-analyzedusing the Jaccard Coefficient (S_(J)) (Sneath, P. H. A. and Sokal, R.R., ibid, p. 131) by running the RTBNSIM program in TAXPAK. A furtherdendrogram was obtained by using the SMATCLST with UPGMA option andDENDGR sub-routines in TAXPAK. A simplified version of this dendrogramis illustrated in FIG. 2. Appendix E gives the percentage positivestates of characters in each cluster.

RESULTS OF THE CLUSTER ANALYSIS S_(G) /UPGMA Method

FIG. 1 shows the results of cluster analysis, based on the GowerCoefficient and the UPGMA method, of 70 new, Gram-negative, alkaliphilicbacteria isolated from in and around alkaline lakes, together with 20known Gram-negative bacteria.

Six natural clusters or phenons of alkaliphilic bacteria which include65 of the 70 alkaliphilic strains are generated at the 73% similaritylevel. Although the choice of 73% for the level of delineation may seemarbitrary, it is in keeping with current practices in numerical taxonomy(Austin, B. and Priest, F., in Modern Bacterial Taxonomy, p. 37; VanNostrand Reinhold; Wokingham, U.K., (1986)). Placing the delineation ata lower percentage would combine groups of clearly unrelated organismswhile a higher percentage would produce a multitude of less well-definedclusters. At the 73% level, the individual clusters may representseparate bacterial genera. Furthermore, the significance of clusteringat this level is supported by chemotaxonomic data (see below) and thepattern of clusters obtained using the Jaccard Coefficient (FIGS. 2A and2B).

The significance of the clustering at the 73% level is supported by theresults of the TESTDEN program. This program tests the significance ofall dichotomous pairs of clusters (comprising 4 or more strains) in aUPGMA generated dendrogram with squared Euclidean distances, or theircomplement, as a measurement. The program assumes that the clusters arehyperspherical. The critical overlap was set at 0.25%. As can be seenfrom Table 5, the separation of the clusters is highly significant.

                  TABLE 5                                                         ______________________________________                                        Significance of the Clusters Generated by the S.sub.G /UPGMA                  Method Provided by TESTDEN                                                    CLUSTER separates from CLUSTER                                                                     at Significance Level                                    ______________________________________                                        1              2         p = 0.99                                             1 + 2          3 + 4     p = 0.99                                             1 + 2 + 3 + 4  5 + 6     p < 0.90                                             1 + 2 + 3 + 4 + 5 + 6                                                                        controls  p = 0.99                                             5              6         p = 0.99                                             ______________________________________                                    

A further measure of cluster separation can be estimated from theprobability of cluster overlap. This was achieved using the OVERMATprogram in TAXPAK with the critical overlap set out at 2.5%. As can beseen from Table 6, there is a greater than 95% probability of less than2.5% overlap between the clusters. For many of the cluster combinationsthe overlap is effectively nil. Only Clusters 3 and 4 have a lowerprobability of <2.5% overlap, but these clusters may be clearlydistinguished from one another on the basis of chemotaxonomic data (seebelow).

                  TABLE 6                                                         ______________________________________                                        Percentage Probability that Cluster Overlap is <2.5%                          CLUSTER  1       2       3     4     5     6                                  ______________________________________                                        2        95                                                                   3        99      95                                                           4        95      95      90                                                   5        99      >99     >99   >99                                            6        >99     >99     99    >99   >99                                      ______________________________________                                    

The controls show that, as expected, the cluster analysis groups theEnterobacteriaceae separately. Additionally, the Aeromonas andPseudomonas species, included as controls, also group separately. Thisis entirely consistent with the current taxonomy of these organisms(Bergey's Manual of Systematic Bacteriology, Volume 1, Williams andWilkins, Baltimore/London, 1984).

Five of the alkaliphilic strains fall outside the major clusters. Twostrains, 4E.1 and 5E.1 form a separate but related pair and areobviously associated with the major groups of alkaliphilic bacteria.Strain wN2 is also unclustered but is apparently related to aPseudomonas species and the major phenons of alkaliphilic bacteria.Strains 92LM.4 and wBn5 do not associate with the major alkaliphilicphenons and probably represent distinct groups of new alkaliphilicbacteria.

Clusters 1 and 2 are the only phenons which show an association withknown organisms, i.e. Pseudomonas and Comamonas species. The separationof Pseudomonas putida and Pseudomonas stutzeri into separate taxa isentirely in keeping with the current taxonomic status of these organisms(Palleroni, N. J. et al, (1973), Int. J. Systematic Bacteriol., 23,333-339; Gavini, F. et al, (1989), ibid, 39, 135-144; Bergey's Manual ofSystematic Bacteriology, supra).

It was clear from the original dendrogram that Pseudomonas stutzeri isan outlier to Cluster 2 and is not closely related to the other membersof the cluster. This is seen when the Euclidean distances of the strainsfrom the centroid of the cluster are computed and used to calculate thecluster radius (Sneath, P. H. A. and Sokal, R. R., supra, pp. 194 etseq). The cluster radius is 3.91 (99% confidence level) and the meandistance of the strains from the centroid is 2.84 (standard deviation0.46). Pseudomonas stutzeri at a distance from the centroid of 3.91 isclearly at the very boundary of phenetic hyperspace which definesCluster 2.

A clear discrimination between Clusters 1 and 2 is possible using theconcept of the minimum discriminatory tests (see below).

Each of the alkaliphilic strains in Cluster 2 have been examined by twoindependent laboratories expert in the identification of bacteria,namely, the German Culture Collection (DSM, Braunschweig, FRG) and theLaboratory for Microbiology at Delft University of Technology, TheNetherlands. Neither of these laboratories was able to make a positiveidentification of the strains, although both agreed there was aresemblance with Pseudomonas either placing them in RNA homology group I(Palleroni, N. J. et al, supra) or more specifically in the Comamonastestosteroni/Pseudomonas alcaligenes or Pseudomonas pseudoalcaligenesgroups (Gavini, F. et al, supra). However, no Pseudomonas species areknown which are able to grow under the same highly alkaline conditions(pH 10) as the new strains described herein. An attempt was made tocultivate Pseudomonas pseudoalcaligenes^(T) DSM 50188 and Pseudomonasalcaligenes^(T) DSM 50342 in an alkaline broth medium (Medium A,Appendix A), but without success.

The results of these experts together with the discoveries describedhere, clearly indicate that these alkaliphilic strains in Clusters 1 and2 represent new species of bacteria.

Clusters 3, 4, 5 and 6 are discrete phenons distinguished from eachother on the basis of the minimum discriminatory tests (see below) andchemotaxonomic markers (see below). These phenons show no significantsimilarity with known groups of bacteria, and thus represent new generaor species.

Whole cell protein patterns generated by PAGE-electrophoresis indicatethat a number of strains are likely to be identical. Examples include:1E.1^(CT) and 2E.1; 6B.1, 7B.1 and 8B.1; 45E.3^(CT) and 47E.3. Thedendrogram reveals that these strains are related at an average S_(G)value of 92.3%, indicating a probable test error of 3.8% (Sneath, P. H.A. and Sokal, R. R., supra). Strains 73bC.4 and 74C.4, which appear tobe closely related (90% S_(G)), have similar but not identical gelpatterns.

S_(J) /UPGMA Method

The Jaccard coefficient is a useful adjunct to the Gower coefficient asit can be used to detect phenons in the latter generated by negativematches or distortions owing to undue weight being put on potentiallysubjective qualitative data. Consequently, the Jaccard coefficient isuseful for confirming the validity of clusters defined initially by theuse of the Gower coefficient. The Jaccard coefficient is particularlyuseful in comparing biochemically unreactive organisms (Austin, B., andPriest, F. G., supra, p. 37).

In the main, all of the clusters generated by the S_(G) /UPGMA methodare recovered in the dendrogram produced by the S_(J) /UPGMA method(FIG. 2). Although the composition of the clusters is virtuallyidentical in both dendrograms, a few strains have changed position.Non-clustering strains 4E.1 and 5E.1 move into Cluster 1/5, strains42E.3 and 50N.3 move from Cluster 2 to Cluster 3/4. The strains wNk2,Pseudomonas stutzeri, Pseudomonas putida, wE5 become non-clustering.

Not surprisingly, the S_(J) transformation combines (S_(G)) Clusters 1and 5. Both of these clusters are characterized as consisting ofbiochemically fairly unreactive strains. However, Clusters 1 and 5 areclearly distinct. Cluster 1 consists of strains producing cream/beige,circular colonies while the strains of Cluster 5 exclusively producebright yellow, irregular colonies.

Furthermore, the S_(J) transformation groups most of the strains ofCluster 4 with the strains of Cluster 3. However, it is evident from thechemotaxonomic data (see below), which shows that the strains of Cluster4 contain Q9 and the strains of Cluster 3 contain mainly Q6, that theseclusters should not be combined since they contain distinctly differentstrains. For these reasons, it is considered that the clusteringproduced by the S_(G) /UPGMA method is the better representation of theactual taxonomic status of these strains. However, the S_(J) /UPGMAserves to re-emphasize that with the single exception of a Comamonasspecies none of the known strains, not even the Pseudomonas controlstrains, bear significant resemblance to the clusters of the newalkaliphilic bacteria.

Chemotaxonomic Definition of the Clusters

Chemotaxonomy is the study of the chemical compositions of organisms inrelation to their systematics. The analysis of chromosomal DNA,ribosomal RNA, proteins, cell walls and membranes, for example, can givevaluable insights into taxonomic relationships and may be used as afurther tool to construct or to verify the taxonomies of microorganisms(Goodfellow, M. and Minnikin, D. E. in Chemical Methods in BacterialSystematics, (eds. Goodfellow, M. and Minnikin, D. E.), Academic Press,London and Orlando, Fla., (1985), pp. 1-15). However, it is not alwayspossible to decide a priori which type of chemical information will bemost diagnostic for a given classification. The amphipathic polarlipids, the major respiratory quinones, fatty acids located in thebacterial membranes and analysis of chromosomal DNA all have taxonomicsignificance for the classification of various bacteria (Lechevalier, H.and Lechevalier, M. P., in Microbial Lipids, volume 1 (eds. Ratledge, C.and Wilkinson, S. G.) Academic Press, London and San Diego, Calif.,(1988), pp. 869-902).

Polar Lipids

The extraction of polar lipids from bacteria and their analysis by twodimensional thin layer chromatography (2D-TLC) may yield patterns ofdiagnostic value. Stationary phase cells were extracted in 1:1 (v/v)CHCl₃ :CH₃ OH and examined by 2D-TLC as described by Ross, H. N. H.,Grant, W. D. and Harris, J. E., in Chemical Methods in BacterialSystematics, (eds. Goodfellow, M. and Minnikin, D. E.), Academic Press,London and Orlando, Fla. (1985), pp. 289-300. The types of lipidspresent on the chromatograms were visualized using a variety ofdifferential stains (Ross, H. N. M., et al., supra, p. 291; andTrincone, A., et al., J. Gen. Microbiol., (1990), 136, pp. 2327-2331).The identity of components were confirmed by co-chromatography withknown lipids.

The results of this analysis for representative strains of Gram-negativealkaliphiles are set out in Table 7. These show no clear polar lipidpattern which is distinct for any one cluster. All strains containphosphatidylglycerol, diphosphatidylglycerol, phosphatidylglycerolphosphate and phosphatidylethanolamine. In addition, certain strains,particularly in Cluster 3, contain phosphatidylglycerol sulphate (PGS).The distribution of PGS within Cluster 3 coincides broadly with thesuspected sub-group structure of the cluster evident from the pheneticand other chemotaxonamic data. PGS is therefore a non-exclusive markerfor Cluster 3.

We were surprised to find that a majority of the bacteria contained aglycolipid which on the basis of numerous co-chromatographic analysesappeared common to Gram-negative bacteria of the present invention.Glycolipids have not previously been demonstrated to be present inalkaliphilic bacteria (Krulwich, T. A., et al, CRC Critical Reviews inMicrobiology, (1988), 16, 15-36). Furthermore, as judged byco-chromatography of lipids obtained from several strains, theglycolipid is also found in Gram-positive alkaliphiles isolated fromsoda lakes. It is possible therefore, that the chemical structure of theglycolipid may be a chemotaxonomic marker for the obligate alkaliphiles.

                                      TABLE 7                                     __________________________________________________________________________    Polar Lipid Components of Gram-Negative Alkaliphilic Bacteria                 CLUSTER                                                                             STRAIN                                                                             PG DPG PGP                                                                              PE  PGS                                                                              GL  AL UPL                                        __________________________________________________________________________    1     1E.1CT                                                                             +  +   +  +                                                              2E.1 +  +   +  +      +                                                       10B.1                                                                              +  +   +  +      3+                                                      20N.1                                                                              +  +   +  +      +                                                       27M.1                                                                              +  +      +      +                                                       wNk.2                                                                              +  +   +  +             +                                          2     39E.3                                                                              +  +   +  +      +                                                       41E.3                                                                              +  +   +  +      +                                                       45E.3.sup.CT                                                                       +  +   +  +      +                                                       51N.3                                                                              +  +   +  +      +                                                       52N.3                                                                              +  +   +  +      +                                                       42E.3                                                                              +  +   +  +      +                                                       50N.3                                                                              +  +   +  +   +                                                          P.s. +  +      +                                                        --    5E.1 +  +   +  +   +  +      +                                          3     6B.1 +  +   +  +          +                                                   7B.1 +  +   +  +      +                                                       8B.1 +  +   +  +      +                                                       25B.1                                                                              +  +   +  +      +                                                       26N.1                                                                              +  +   +  +      +                                                       12C.1                                                                              +  +   +  +      +   +                                                   28N.1.sup.CT                                                                       +  +   +  +   +                                                          36E.2                                                                              +  +   +  +   +  +                                                       40E.3                                                                              +  +   +  +   +  +                                                       94LM.4                                                                             +  +   +  +   +  +                                                       19N.1                                                                              +  +   +  +      +                                                       24B.1                                                                              +  +   +  +      +                                                       21M.1                                                                              +  +   +  +      +                                                       29C.1                                                                              +  +   +  +   +                                                          35E.2                                                                              +  +   +  +   +                                                          37E.2                                                                              +  +   +  +   +  +                                                       48E.3                                                                              +  +   +  +   +  +                                                       73aC.4                                                                             +  +   +  +   +  +                                                       74C.4                                                                              +  +   +  +   +  +                                                       49E.3                                                                              +  +   +  +   +  +                                                       44E.3                                                                              +  +   +  +   +  +                                                       58E.4                                                                              +  +   +  +      +                                                 4     wE5  +  +   +  +   +                                                          wB4.sup.CT                                                                         +  +   +  +                                                              wNk1 +  +   +  +          +  +                                                wEl1 +  +   +  +          +  +                                                wEl2 +  +   +  +             +                                          5     9B.1 +  +   +  +                                                              16N.1                                                                              +  +   +  +                                                              17N.1.sup.CT                                                                       +  +   +  +      +                                                       22M.1                                                                              +  +   +  +                                                        6     18N.1                                                                              +  +   +  +      +                                                       59E.4                                                                              +  +   +  +                                                              64B.4.sup.CT                                                                       +  +   +  +                                                              63N.4                                                                              +  +   +  +      +                                                       53E.4                                                                              +  +   +  +      +                                                 __________________________________________________________________________     (PG) phosphatidylglycerol;                                                    (DPG) diphosphatidylglycerol;                                                 (PGP) phosphatidylglycerol phosphate;                                         (PE) phosphatidylethanolamine;                                                (PGS) phosphatidylglycerol sulphate;                                          (GL) unidentified glycolipid(s), α-naphthol positive (the number in     the column gives the number of positive spots on the TLC plate);              (AL) unidentified aminolipid (ninhydrin positive);                            (UPL) unidentified phospholipid(s).                                      

Isoprenoid Quinones

The isoprenoid or respiratory quinones are characteristic components ofthe plasma membrane of aerobic bacteria. There are two types;menaquinones and ubiquinones. The value of isoprenoid quinones astaxonomic criteria lies in the variation in the length of the polyprenylside-chain and the degree of saturation (Collins, M. D. and Jones, D.(1981), supra).

Freeze dried stationary phase bacterial cells were extracted, using amodified procedure of Collins, M. D. (in Chemical Methods in BacterialSystematics, supra, pp. 267-284), in 1:1 (v/v) CHCl₃ :CH₃ OH at 50° C.,for 16 hours. The quinones were examined by reverse phase thin layerchromatography as described by Collins, M. D. (supra).

The results of quinone analyses of nearly all the strains ofGram-negative alkaliphiles are illustrated in Table 8. All of thestrains tested contained exclusively ubiquinones which confirms theirstatus as Gram-negative bacteria (Collins, M. D. and Jones, D., supra).Table 8 shows quite clearly that the major ubiquinones are Q6 and Q9. Itis also evident that the strains containing Q6 are exclusive to Cluster3 and that this distinguishes Cluster 3 from all the other clusterssince they contain strains possessing Q9 as the major ubiquinone.

                                      TABLE 8                                     __________________________________________________________________________    Major Respiratory Quinones of the Strains Arranged per Cluster                __________________________________________________________________________    CLUSTER 1     CLUSTER 2   CLUSTER 3                                           STRAIN                                                                              Q       STRAIN                                                                              Q     STRAIN                                                                              Q                                             __________________________________________________________________________    1E.1.sup.CT                                                                         Q9      39E.3 Q9    6B.1  Q6                                            2E.1  Q9      41E.3.sup.CT                                                                        Q9    7B.1  Q6, Q9                                        wB2   Q9      45E.3 Q9    8B.1  Q6, Q9                                        wB5   Q9      47E.3 Q9, Q10                                                                             38E.2 Q6                                            wBs4  Q9      52N.3 Q9    56E.4 Q6                                            10B.1 Q9      42E.3 Q9    25B.1 Q6, Q9                                        20N.1 Q9      50N.3 Q9    26N.1 Q6                                            27M.1 Q9                  12C.1 Q6                                            C.t.* [Q8 + Q9(t)]        28N.1.sup.CT                                                                        Q6                                            wNk2  Q9                  61N.4 Q6                                            P.p.* [Q9]                36E.2 Q6                                                                      40E.3 Q6, Q9                                                                  65B.4 Q6                                                                      94LM.4                                                                              Q6, Q9                                                                  19N.1 Q6, Q9                                                                  24B.1 Q6, Q9                                                                  21M.1 Q6                                                                      29C.1 Q6                                                                      35E.2 Q6                                                                      37E.2 Q6                                                                      48E.3 Q6                                                                      78LN.4                                                                              Q6                                                                      73aC.4                                                                              Q6                                                                      75C.4 Q6                                                                      73bC.4                                                                              Q6                                                                      74C.4 Q6, Q9                                                                  77LN.4                                                                              Q6                                                                      49E.3 Q6                                                                      58E.4 Q6                                                                      57E.4 Q6                                            __________________________________________________________________________    CLUSTER 4  CLUSTER 5 CLUSTER 6                                                                            NON-CLUSTER                                       STRAIN                                                                             Q     STRAIN                                                                             Q    STRAIN                                                                             Q STRAIN                                                                             Q                                            __________________________________________________________________________    wE5.sup.CT                                                                         Q9    9B.1 Q9   18N.1                                                                              Q9                                                                              wN.2 Q9                                           wB4  Q9    16N.1                                                                              Q9   59E.4.sup.CT                                                                       Q9                                                                              5E.1 Q8                                           wNk1 Q9    17N.1.sup.CT                                                                       Q9,  64B.4                                                                              Q9                                                                              92LM.4                                                                             Q9                                                           Q10                                                           wEl1 Q9, Q10(t)                                                                          22M.1                                                                              Q9   63N.4                                                                              Q9                                                                              wBn5 Q8, Q9(t)                                    wEl2 Q9    22M.1                                                                              Q9   53E.4                                                                              Q9                                                  __________________________________________________________________________     Q = Ubiquinone, the number indicates the number of sidechain isoprene         units.                                                                        (t) = trace                                                                   *C.t. = Comamonas terrigena.sup.T NCIMB 8193, the quinone result is           obtained from J. Tamaoka et al, International Journal of Systematic           Bacteriology, 37, 52-59, (1987).                                              P.p. = Pseudomonas putida.sup.T NCIMB 9494, the quinone result is obtaine     from M. D. Collins and D. Jones, Microbiological Reviews, 45, 316-354,        (1981).                                                                  

Fatty Acids

The analysis of fatty acid profiles has had a significant impact onbacterial classification especially in the circumscription of genera andspecies among Gram-positive bacteria and actinomycetes (Kroppenstedt, R.M., in Chemical Methods in Bacterial Systematics (eds. M. Goodfellow andD. E. Minnikin), Academic Press; London and Orlando, Fla., (1985), pp.173-199); Lechevalier, H. and Lechevalier, M. P., supra.

Freeze dried stationary phase cells (200-300 mg) were extracted for 16hours at 75° C. in toluene:methanol:conc. sulphuric acid (2.5 ml:2.5ml:0.2 ml) and after cooling, the lipids were partitioned into hexane(twice times 1 ml). Residual acid was removed using NH₄ HCO₃. Lipidextracts were concentrated under O₂ -free N₂, dissolved in 300 μl hexaneand applied to preparative silica gel plates (Merck F254, Type T). Theplates were developed in hexane: diethyl ether 85:15 (v/v) and the fattyacid methyl esters scraped off, extracted with hexane and concentratedunder a stream of O₂ -free N₂.

The fatty acid methyl esters were dissolved in heptane and analyzed bygas chromatography using a Packard model 439 chromatograph equipped withflame ionization detectors. The samples were divided by a samplesplitter and analyzed simultaneously over two columns, namely, CP-SIL-88(Chrompack) (length 50 meter, internal diameter 0.22 mm) and Ultra-2(Hewlett/Packard) (length 50 m, internal diameter 0.20 mm). The carriergas was nitrogen; the injection temperature 120° C.; temperaturegradient 2.5° C. per minute to 240° C. and isothermal at 240° C. for 30minutes. Fatty acid methyl esters were assigned by reference to knownstandard mixtures. The identity of some peaks was confirmed by means ofgas chromatography-mass spectrometry using a Carlo Erba HRGC 5160 Megaseries gas chromatograph equipped with a CP-SIL-88 column (length 50meter, internal diameter 0.22 mm) with helium as carrier gas and directinjection into the source of a AMD 403 mass spectrometer.

The fatty acid compositions of representative individual Gram-negativebacteria are set out in Table 9. Table 10 shows the unique fatty acidprofiles of each of the clusters. Clusters 1, 2, 3 and 4 are fairlytypical of the majority of Gram-negative bacteria where the majorsaturated fatty acid is C16:0 with lesser amounts of C14:0 and C18:0.The major unsaturated fatty acids in these alkaliphilic bacteria areC16:0 and C18:1 (11-cis), which is also typical, as is the lack ofodd-numbered fatty acids (Wilkinson, S. G., in Microbial Lipids, volume1 (eds. Ratledge, C. and Wilkinson, S. G.), Academic Press, London andSan Diego, Calif., (1988), pp. 299-488). Minor amounts of C17:0 andC19:0 cyclopropane acids are found in some strains of Gram-negativebacteria. The strains of Cluster 3 exhibit fairly simple fatty acidprofiles with C16:0 and C18:1 contributing 67-88% of the total acids,and C16:1 plus C18:0 up to 20% of the remainder. Even so, the fatty acidpatterns support the notion that Cluster 3 contains several sub-groups,a conclusion that is also inferred from phenetic (numerical taxonomy)and polar lipid analyses (Table 7).

The strains of Cluster 1 can be distinguished from those of Cluster 2 onthe relative abundance of straight chain saturated and unsaturated fattyacids, as well as the percentage amounts of C18:1(11-cis). Thealkaliphilic bacteria of Clusters 1 and 2 have more complex fatty acidprofiles than those of Cluster 3, with many more minor components. Fromthe numerical taxonomy evidence, the alkaliphilic strains of Clusters 1and 2 exhibit some resemblance to Pseudomonas species. However, thetotal lack of any hydroxy-fatty acids which are typical of mostPseudomonas species, further indicating that a close relationship isdoubtful.

The strains of Clusters 5 and 6 are remarkable in that besidescontaining major amounts of C16:0, the other major fatty acids areodd-numbered branched chain acids (40-85%). Also, these strains lacksignificant amounts of C18:1 or any other unsaturated acids which arepresent in appreciable amounts in the alkaliphilic strains of Clusters1, 2, 3 and 4. The presence of large amounts of C15:0 and C17:0 iso andanteiso acids is characteristic of only a very few classes ofGram-negative bacteria, notably species from exotic environments such asThermus, or poorly defined taxa such as Flavobacterium (Wilkinson, S.G., supra). This result further emphasizes the novelty of thealkaliphilic strains of the present invention. The strains of Clusters 5and 6 can be distinguished from each other by the proportion ofbranched-chain fatty acids they contain and more especially by therelative proportions of even- and odd-numbered fatty acids.

                  TABLE 9                                                         ______________________________________                                        Fatty Acid Composition.sup.+ of Gram-Negative Alkaliphiles                    ______________________________________                                        CLUSTER →                                                                           1                2                                               FATTY ACID   1E.1.sup.CT                                                                           2E.1     45E.3.sup.CT                                                                         50E.3                                    ______________________________________                                        10:0                                                                          12:0         t       t        3.6    4.7                                      12:1         0.2     0.3                                                      13:0                          <0.1                                            14:0         0.7     0.7      3.8    3.9                                      14:0 iso                                                                      14:1         2.6     0.9                                                      15:0                 0.3      0.6    0.9                                      15:0 iso                      0.1    0.2                                      15:0 anteiso                  <0.1   0.3                                      15:0 cyclo                                                                    16:0         29.0    32.0     28.5   34.4                                     16:0 iso             0.3             0.2                                      16:1         7.4     9.6      4.1    4.3                                      17:0         0.5     2.3      0.9    1.1                                      17:0 iso                      0.2    0.6                                      17:0 anteiso                                                                  17:0 cyclo                    0.4.sup.a                                                                            1.8                                      17:1         0.3     1.6                                                      17:1 br                              1.7                                      18:0         12.0    4.7      17.9   10.8                                     18:0 unknown 0.2     0.4                                                      18:1 9-cis   0.2     t        0.5    0.3                                      18:1 9-trans 0.5     0.6      5.9    2.9                                      18:1 11-cis  42.0    44.0     23.9   24.1                                     18:1 unknown 0.2     0.3                                                      18:2                          0.5    1.9                                      19:0                          0.1                                             19:0 cyclo                           1.3                                      19:1 br                                                                       20:0         0.4              5.3    2.7                                      20:1         3.6     1.2                                                      22:0                          3.3    1.5                                      24:0                          0.2                                             ______________________________________                                        CLUSTER                                                                       →                                                                      FATTY   3                                                                     ACID    25B.1   28N.1.sup.CT                                                                           36E.2 24B.1 37E.2 48E.3                              ______________________________________                                        10:0            0.4                                                           12:0            0.5      0.5         0.3   t                                  12:1                                                                          13:0                                                                          14:0    4.5     4.4      3.4   2.6   2.9   2.6                                14:0 iso                                                                      14:1            0.1                                                           15:0    0.7     0.6                  0.9   0.3                                15:0 iso                                                                      15:0 anteiso                                                                  15:0 cyclo                                                                    16:0    37.3    24.1     42.0  36.7  33.3  26.0                               16:0 iso                                                                      16:1    11.6    15.0     10.0  5.8   3.4   8.0                                17:0            0.2                  1.2   0.3                                17:0 iso                                                                      17:0 anteiso                                                                  17:0 cyclo                           1.8   0.3                                17:1    0.6                                                                   17:1 br                                                                       18:0    8.9     0.2      0.9   2.0   0.6   1.1                                18:0                                                                          unknown                                                                       18:1 9-cis      t        t     t     0.6   t                                  18:1 9-trans                                                                          2.4     0.2      t     1.0   0.6   0.6                                18:1 11-cis                                                                           27.5    54.2     43.0  50.6  41.6  57.7                               18:1                                                                          unknown                                                                       18:2    2.7                    0.5   t     t                                  19:0                                                                          19:0 cyclo      0.4                  12.9  2.0                                19:1 br                                                                       20:0    2.4                                                                   20:1                                                                          22:0    1.4                                                                   24:0                                                                          ______________________________________                                        CLUSTER →                                                                         4            5                                                     FATTY ACID WB4.sup.CT                                                                             WEl1    9B.1  16N.1 17N.1.sup.CT                          ______________________________________                                        10:0       0.5                                                                12:0       0.9      0.2                                                       12:1                0.8                                                       13:0                                                                          14:0       3.8      1.3     3.2   1.9   1.2                                   14:0 iso                    0.7   t     t                                     14:1                0.1                                                       15:0       0.7      0.2                                                       15:0 iso                    8.7   9.3   8.2                                   15:0 anteiso                32.3  27.1  35.3                                  15:0 cyclo <0.1                                                               16:0       26.8     26.9    22.5  17.4  12.5                                  16:0 iso                    4.7   5.8   6.7                                   16:1       7.9      2.4                                                       17:0       0.4      0.3                                                       17:0 iso            0.3     3.2   6.0   5.1                                   17:0 anteiso                21.9  24.4  29.8                                  17:0 cyclo 0.2                                                                17:1       <0.1                                                               17:1 br                                                                       18:0       5.2      3.5     2.3   5.3   1.2                                   18:0 unknown                                                                  18:1 9-cis t        2.5                                                       18:1 9-trans                                                                             1.7      0.9     *0.5  *1.6  *1.0                                  18:1 11-cis                                                                              47.4     48.1                                                      18:1 unknown                                                                  18:2       1.0      0.3                                                       19:0                                                                          19:0 cyclo 1.0                                                                19:1 br             11.2                                                      20:0       1.3      0.4                                                       20:1                0.3                                                       22:0       0.8      0.2                                                       24:0                                                                          ______________________________________                                        CLUSTER →                                                                         6              non-clustering                                      FATTY ACID 59E.4    64B.4.sup.CT                                                                            5E.1   92LM.4                                   ______________________________________                                        10:0                                                                          12:0                                                                          12:1                                                                          13:0                                                                          14:0       3.9      6.4       3.8    1.5                                      14:0 iso   0.7      2.0                                                       14:1                                                                          15:0       t        3.8       t                                               15:0 iso   3.9      12.6             44.0                                     15:0 anteiso                                                                             24.8     18.1             9.9                                      15:0 cyclo                                                                    16:0       26.3     40.5      74.7   18.7                                     16:0 iso   2.4      3.7              2.1                                      16:1                          2.9                                             17:0       t        2.4                                                       17:0 iso   0.6      1.7              15.7                                     17:0 anteiso                                                                             6.1      3.7              6.8                                      17:0 cyclo                                                                    17:1                                                                          17:1 br                                                                       18:0       16.2     4.8       2.6    1.5                                      18:0 unknown                  5.9                                             18:1 9-cis                                                                    18:1 9-trans                                                                             *5.7     *0.5      *10.0                                           18:1 11-cis                                                                   18:1 unknown                                                                  18:2       2.7                                                                19:0                                                                          19:0 cyclo                                                                    19:1 br                                                                       20:0       4.6                                                                20:1                                                                          22:0       2.6                                                                24:0                                                                          ______________________________________                                         * = includes all C18:1 isomers                                                + = % total fatty acids                                                       t = trace                                                                     br = branched                                                                 a = C17:0 cyclo or C18:0 unknown                                         

                  TABLE 10                                                        ______________________________________                                        Fatty Acid Profiles of the Clusters of Gram-Negative Alkaliphiles             ______________________________________                                                Cluster                                                                         1         2         3A/B   3C                                       ______________________________________                                        Predominant                                                                             C16:0     C16:0     C16:0  C16:0                                    Fatty Acids                                                                             C18:1 11-cis                                                                            C18:0     C16:1  C18:1 11-cis                             (>10%)              C18:1 11-cis                                                                            C18:1                                                                         11-cis                                          n-saturated                                                                             ˜40%                                                                              60-65%    30-55% ˜40%                               n-unsaturated                                                                           ˜60%                                                                              ≈33%                                                                            45-70% ˜60%                               iso       <1%       <1%       0%     0%                                       anteiso   0%        <1%       0%     0%                                       total branched                                                                          <1%       <3%       0%     0%                                       cyclo     0%        <5%              0%                                       even carbon no.                                                                         >95%      >90%      >99%   >99%                                     odd carbon no.                                                                          <5%       <10%      <1%    <1%                                      additional          C17:0 cyclo                                               markers             C19:0 cyclo                                                                   C17:1 br                                                  ______________________________________                                                Cluster                                                                         3D        4         5      6                                        ______________________________________                                        Predominant                                                                             C16:0     C16:0     C15:0  C15:0 br                                 Fatty Acids                                                                             C18:1 11-cis                                                                            C18:1 11-cis                                                                            anteiso                                                                              C16:0                                    (>10%)                        C16:0                                                                         C17:0                                                                         anteiso                                         n-saturated                                                                             30-40%    30-40%    15-30% 55-60%                                   n-unsaturated                                                                           50-65%    50-60%    <2%    <10%                                     iso       0%        <1%       ˜20%                                                                           10-20%                                   anteiso   0%        0%        50-65% 20-30%                                   total branched                                                                          0%        1-12%     >70%   ˜40%                               cyclo     2-15%     <2%       0%     0%                                       even carbon no.                                                                         >80%      >85%      20-35% 55-65%                                   odd carbon no.                                                                          <20%      <15%      65-80% 35-45%                                   additional                                                                              C17:0 cyclo                                                         markers   C19:0 cyclo                                                         ______________________________________                                         br = branched                                                            

Nucleic Acids

An essential component of any taxonomic study is an analysis of thegenetic material--the nucleic acids. The composition of chromosomal DNAis unaffected by the growth conditions of the organism and anappropriate analysis may confirm or refute the taxonomic position of theorganism. Chromosomal DNA may be analyzed by the determination of thebase composition (G+C mol %) of individual strains, and the basesequence homologies between pairs of strains by DNA--DNA reassociation(hybridization) (Owen, R. J. and Pitcher, D., in Chemical Methods inBacterial Systematics (eds. M. Goodfellow and D. E. Minnikin), AcademicPress, London and Orlando, Fla. (1985), pp. 67-93).

DNA Base Composition

The guanine plus cytosine (G+C mol %) composition is constant for thechromosomal DNA from any given organism. Closely related organisms havesimilar G+C compositions. However, G+C results must be interpretedwithin the context of independent taxonomic data since similar G+C mol %of DNA samples from different organisms does not in itself implybiological relatedness.

DNA was extracted from cells grown to exponential phase in Medium A bythe chloroform:phenol method and was precipitated with ethanol. Basecomposition was determined by the thermal denaturation method (Marmur,J. and Doty, P. (1962), J. Mol. Biol., 3, 585-594) on a Phillips modelPV8764 spectrophotometer with temperature programming. A second methodinvolved HPLC analysis on a Beckman system gold using a Beckmanultrasphere ODS column and 0.04M potassium dihydrogen phosphate plusacetonitrile (9+1, v/v) as eluent at a flow rate of 1.5 ml/min, aftertreatment of the DNA with nuclease P1 and alkaline phosphatase.

The results of these analyses are set out in Table 11. The G+C mol %values for the alkaliphilic bacteria cover a range of 30 mol %(37.6-67.1 mol %). However, within the clusters the variation is only3-7 mol %, which further confirms that the strains within a cluster areclosely related to each other.

                  TABLE 11                                                        ______________________________________                                        DNA Base Composition of Gram-Negative                                         Alkaliphilic Bacteria                                                                           G + C mol %                                                 Cluster      Strain     HPLC    T.sub.M                                       ______________________________________                                        1            2E.1       55.2                                                               wBs4               51.2                                                       20N.1      51.1                                                               wNk2               53.0                                          2            42E.2      62.7                                                  3            wB1                63.0                                                       28N.1.sup.CT                                                                             64.1                                                               37E.2      67.1                                                               wN1                64.8                                          4            wE5                58.5                                                       wB4.sup.CT         65.3                                                       wNk1               61.0                                                       wEl1               59.7                                                       wEl2               58.1                                          5            17N.1.sup.CT                                                                             50.0                                                               22M.1      43.8                                                  6            64B.4.sup.CT                                                                             41.0                                                               53E.4      37.6                                                  non          wN2                64.1                                                       wBn5               54.6                                          ______________________________________                                    

DNA--DNA Molecular Hybridization

The method used was essentially that of Crosa, J. H. et al. (Int. J.Systematic Bacteriol., 29, 328-332, 1979). Tritium labelled DNA wasprepared using a nick-translation kit (Amersham, N5000) according to themanufacturer's instructions. The reassociation mixtures were incubatedat 65° C. for 16 hours. The results are set out in Table 12 from whichit can be seen that the DNA sequence homology is higher within theclusters that between the clusters.

                                      TABLE 12                                    __________________________________________________________________________    Inter-Cluster and Intra-Cluster DNA-DNA Homology                              Values for Gram-Negative Alkaliphilic Bacteria                                         1  2    3    4   5    6                                              Cluster                                                                           Strain                                                                             2E.1                                                                             45E.3.sup.CT                                                                       28N.1.sup.CT                                                                       wEl2                                                                              17N.1.sup.CT                                                                       64B.4.sup.CT                                   __________________________________________________________________________    1   2E.1 100     33   35  25   25                                                 20N.1                                                                              56                                                                   2   45E.3.sup.CT                                                                          100       25                                                          42E.3   76            30                                                  3   28N.1.sup.CT                                                                       20 34   100  26  30   30                                                 56E.4        51                                                               21M.1        53                                                               37E.2   37                                                                    44E.3        55                                                           4   wEl2              100                                                         wB4.sup.CT   41   43                                                      5   17N.1.sup.CT                                                                       45 44   36   24  100  44                                                 22M.1                 65                                                  6   64B.4.sup.CT                                                                       21 31   35   25  34   100                                                53E.4                      60                                                 SS   17               20   20                                             __________________________________________________________________________     The values give the percent hybridization between the strains (rows) and      H.sup.3 -labelled strains (columns).                                          SS = salmon sperm DNA.                                                   

Determination of Representative Strains

The centroid of each individual cluster generated by the S_(G) /UPGMAmethod was computed using the RGROUPS program in TAXPAK. The centroid ofa cluster of points representing real organisms projected intohyperspace represents a hypothetical average organism. The centroidrarely, if ever represents a real organism. Therefore, the Euclideandistances of each of the members of the cluster from the centroid of thecluster were calculated in order to establish which strain was closestto the hypothetical average organism. The strain closest to the centroidwas designated the "centrotype" organism (indicated with the superscript"CT").

The centrotype organism can be thought of as the "Type Strain" whichmost closely represents the essential and discriminating features ofeach particular cluster. The centrotype strains are recorded in Table13.

                  TABLE 13                                                        ______________________________________                                        Centrotype Strains                                                                     Mean                                                                          Euclidean         Centrotype                                               Number   Distance of             Euclidean                              Cluster                                                                             of       Strains                 Distance                               Num-  Strains in                                                                             from      Standard      from                                   ber   Cluster  Centroid  Deviation                                                                             Strain                                                                              Centroid                               ______________________________________                                        1     11       3.67      0.30    1E.1  2.88                                   2     9        3.20      0.52    45E.3 2.30                                   3     34       3.52      0.32    28N.1 2.90                                   4     5        3.97      0.29    wB4   2.87                                   5     4        3.25      0.29    17N.1 2.13                                   6     5        3.11      0.41    64B.4 1.93                                   ______________________________________                                    

A description of each of the centrotype organisms has been made so as tobe able to distinguish these organisms from all other bacteriapreviously known and described. In addition, the minimum number ofdiscriminating tests to define each cluster has been computed so that itmay be clearly seen that the clusters containing these novel bacteriacan be easily distinguished from each other and from all other knownbacteria.

DESCRIPTION OF CENTROTYPE STRAINS Strain 1E.1^(CT) (Cluster 1)

An aerobic, motile, Gram-negative rod-shaped bacterium, 1.7-3.3μm×0.5-0.7 μm.

Obligate alkaliphile, grows best between pH 9 and pH 10.

On alkaline-agar, (Medium A) forms smooth, cream colored colonies,initially translucent but becoming opaque after a few days. The coloniesare circular, entire and convex, 2-3 mm in diameter.

In alkaline-broth, (Medium A) growth (37° C.) is flocculent with theformation of a sediment and surface pellicle.

Grows well between 20° C. and 40° C. Grows slowly at 10°-15° C. Nogrowth at 8° C. or 45° C.

    ______________________________________                                        KOH test:        positive                                                     Aminopeptidase:  weak positive                                                Oxidase:         negative                                                     Catalase:        positive                                                     NaCl tolerance:  0% to <8%. No growth at 8%                                   Hydrolysis of Gelatin:                                                                         positive                                                     Hydrolysis of Starch:                                                                          positive                                                     Major polar lipid components:                                                                  phosphatidylglycerol                                                          diphosphatidylglycerol                                                        phosphatidylglycerol phosphate                                                phosphatidylethanolamine                                     Major ubiquinone:                                                                              Q9                                                           Major fatty acids:                                                                             C16:0, C18:0, 11-cis C18:1                                   ______________________________________                                    

Chemoorganotroph. Grows on complex substrates such as yeast extract andpeptones. Growth on simple sugars and organic acids very restricted(e.g., growth only observed on ribose, sucrose and pyruvate).

Strain 45E.3^(CT) (Cluster 2)

An aerobic, Gram-negative, rod-shaped bacterium, 3-4.5 μm×0.6 μm. Motileby a single polar flagellum.

Obligate alkaliphile growing between pH 7.8 and pH 11.2. O nalkaline-agar, (Medium A) forms smooth, opaque, cream colored colonies,1-2 mm in diameter. The colonies are circular, convex and entire.

In alkaline-broth, (Medium A) growth (37° C.) is slow, slight with aneven turbidity, surface pellicle and no sediment.

Grows well between 20° C. and 40° C. Grows slowly at 10° C. No growth at8° C. or 45° C.

    ______________________________________                                        KOH test:        positive                                                     Aminopeptidase:  positive                                                     Oxidase:         positive                                                     Catalase:        positive                                                     NaCl tolerance:  0% to 12%. Growth at 12% is                                                   slow                                                                          No growth at 15%                                             Hydrolysis of Gelatin:                                                                         positive                                                     Hydrolysis of Starch:                                                                          positive (weak)                                              Major polar lipid components:                                                                  phosphatidylglycerol                                                          diphosphatidylglycerol                                                        phosphatidylglycerol phosphate                                                phosphatidylethanolamine                                                      glycolipid (α-naphthol positive)                       Major ubiquinone:                                                                              Q9                                                           Major fatty acids:                                                                             C16:0, C18:0, 11-cis C18:1                                   ______________________________________                                    

Chemoorganotroph. Grows on complex substrates such as yeast extract andpeptones. No growth on simple sugars. Grows on organic acids (e.g.,fumarate, succinate, pyruvate, acetate, lactate) and some fatty acids(e.g., propionate, valerate) and amino acids (e.g., proline, alanine,phenylalanine).

Strain 28N.1^(CT) (Cluster 3)

An aerobic, motile, Gram-negative, rod-shaped bacterium, 4.8-5.5μm×0.6-0.8 μm. Obligate alkaliphile growing between pH 8.5 and pH 10.7.

On alkaline-agar, (Medium A) forms smooth, circular, opaque colonieswith a stringy texture. The colonies have a convex elevation and entiremargin. The colony color is initially cream/beige becoming pink after afew days.

In alkaline-broth, (Medium A) growth (37° C.) is heavy, flocculent witha surface pellicle and a sediment.

Grows well between 20° C. and 45° C. Grows slowly at 10° C. and 15° C.No growth at 50° C.

    ______________________________________                                        KOH test:        positive                                                     Aminopeptidase:  positive                                                     Oxidase:         positive                                                     Catalase:        positive                                                     NaCl tolerance:  0% to 12%. No growth at 15%                                  Hydrolysis of Gelatin:                                                                         negative                                                     Hydrolysis of Starch:                                                                          positive                                                     Major polar lipid components:                                                                  phosphatidylglycerol                                                          diphosphatidylglycerol                                                        phosphatidylglycerol phosphate                                                phosphatidylglycerol sulphate                                                 phosphatidylethanolamine                                     Major ubiquinone:                                                                              Q6                                                           Major fatty acids:                                                                             C16:0, C16:1, 11-cis C18:1                                   G + C:           64.1 mol % (HPLC)                                            ______________________________________                                    

Chemoorganotroph. Grows well on complex substrates such as yeast extractand peptones. Grows on simple sugars, organic acids, fatty acids andamino acids.

Strain wB4^(CT) (Cluster 4)

An aerobic, Gram-negative, rod-shaped bacterium, 3-4 μm×0.6-0.8 μm,frequently occurring as pairs of cells.

Alkaliphile, grows well between pH 7.5 and pH 10.9.

On alkaline-agar, (Medium A) forms smooth, beige to brown colonies. Thecolonies are somewhat variable: 1 to >5 mm in size, circular toirregular in form, low convex or raised in elevation with an undulate orentire margin.

In alkaline broth, (Medium A) growth (37° C.) is flocculent, sedimentforming with a surface pellicle.

Grows best between 15° C. and 45° C., no growth at 50° C.

    ______________________________________                                        KOH test:        positive                                                     Aminopeptidase:  positive                                                     Oxidase:         very weakly positive, may be                                                  seen as negative                                             Catalase:        positive                                                     NaCl tolerance:  0% to ≧12%, no growth at 15%                          Hydrolysis of Gelatin:                                                                         negative                                                     Hydrolysis of Starch:                                                                          negative                                                     Major polar lipid components:                                                                  phosphatidylglycerol                                                          diphosphatidylglycerol                                                        phosphatidylglycerol phosphate                                                phosphatidylethanolamine                                     Major ubiquinone:                                                                              Q9                                                           Major fatty acids:                                                                             C16:0, 11-cis C18:1                                          G + C:           65.3 mol % (T.sub.M)                                         ______________________________________                                    

Chemoorganotroph. Grows well on complex substrates such as yeastextract. Growth on simple sugars is restricted. Grows on organic acids(e.g., lactate, acetate, fumarate), fatty acids (e.g., propionate,valerate, caprate) and amino acids (e.g., proline, serine, lysine).

Strain 17N.1^(CT) (Cluster 5)

An aerobic, Gram-negative, long, thin, rod-shaped, bacterium, 5.5-10.5μm×0.6 μm, sometimes forming short chains of cells. With agepleomorphic, peculiar swollen forms predominate.

Obligate alkaliphile, grows best between pH 8 and pH 10.5.

On alkaline-agar, (Medium A) forms smooth, opaque, yellow, colonies, 2-3mm in diameter. The colonies vary from circular to irregular in form,with a convex to umbonate elevation, and entire, undulate or lobatemargin, depending upon age.

In alkaline-broth (Medium A), growth (37° C.) is even and sedimentforming with no surface pellicle.

Grows well between 15° C. and 37° C. Grows slowly at 10° C. and not atall at 8° C. No growth at 40° C. or above.

    ______________________________________                                        KOH test:        positive                                                     Aminopeptidase:  weakly positive                                              Oxidase:         negative                                                     Catalase:        positive                                                     NaCl tolerance:  0% to <12%, grows best at                                                     0% NaCl                                                      Hydrolysis of Gelatin:                                                                         positive                                                     Hydrolysis of Starch:                                                                          weakly positive                                              Major polar lipid components:                                                                  phosphatidylglycerol                                                          diphosphatidylglycerol                                                        phosphatidylglycerol phosphate                                                phosphatidylethanolamine                                                      glycolipid (α-naphthol positive)                       Major ubiquinone:                                                                              Q9, Q10                                                      Major fatty acids:                                                                             C15:0 anteiso, C16:0, C17:0                                                   anteiso                                                      G + C:           50.0 mol % (HPLC)                                            ______________________________________                                    

Chemoorganotroph. Grows well on complex substrates such as yeastextract. Growth on simple sugars is restricted (e.g., growth observedonly on fructose; no growth observed on glucose, ribose, lactose). Growson organic acids (e.g., fumarate, succinate, pyruvate, 2-ketogluconate)and amino acids.

Strain 64B.4^(CT) (Cluster 6)

An aerobic, Gram-negative, rod-shaped bacterium 2.0-3.5 μm×0.8-1.0 μm.

Obligate alkaliphile, grows best between pH 8.2 and pH 10.9.

On alkaline-agar, (Medium A) forms smooth, opaque colonies, first creamyyellow in color, becoming beige with age. The colonies are about 4 mm indiameter, circular becoming irregular; flat or low convex in elevationbecoming convex; with an entire margin becoming undulate.

In alkaline-broth (Medium A), growth (37° C.) is even, sediment formingwith no surface pellicle.

Grows well at 15° C. to 45° C., no growth at 10° C. or 50° C.

    ______________________________________                                        KOH test:        positive                                                     Aminopeptidase:  negative                                                     Oxidase:         positive                                                     Catalase:        positive                                                     NaCl tolerance:  0% to ≦12%, no growth at 15%                          Hydrolysis of Gelatin:                                                                         positive                                                     Hydrolysis of Starch:                                                                          weakly positive                                              Major polar lipid components:                                                                  phosphatidylglycerol                                                          diphosphatidylglycerol                                                        phosphatidylglycerol phosphate                                                phosphatidylethanolamine                                                      glycolipid (α-naphthol positive)                       Major ubiquinone:                                                                              Q9                                                           Major fatty acids:                                                                             C15:0 iso, C15:0 anteiso, C16:0                              G + C:           41.0 ± 0.9 mol % (HPLC)                                   ______________________________________                                    

Chemoorganotroph. Grows well on complex substances such as yeastextract. Grows on some simple sugars (e.g., glucose, ribose, maltose andfructose), organic acids (e.g., acetate, lactate, citrate and fumarate),some fatty acids (e.g., propionate and caprate) and amino acids (e.g.,proline, histidine and alanine).

Non-Clustering Strains

The strains which do not fall into the clusters defined here are alsonovel bacteria not previously known or described. These strains, codedwN2, 4E.1, 5E.1, 92LM.4 and wBn5, may represent rarer varieties ofalkaliphilic bacteria and are probably members of clusters of bacteriarepresenting new genera and species at present not described. Adescription of these "non-clustering" strains has been made so as to beable to distinguish these organisms from all other bacteria previouslyknown and described.

Strain wN2

An aerobic, Gram-negative, motile, rod-shaped bacterium, frequently inpairs.

Obligate alkaliphile, grows best between pH 9 and pH 10.

On alkaline-agar, (Medium A) forms smooth, translucent, beige coloredcolonies, 1-2 mm in diameter. The colonies are circular, convex with anentire margin.

In alkaline-broth (Medium A), growth (37° C.) is flocculent with a ringor surface pellicle and formation of a sediment.

Grows well at 20° C. to 30° C. No growth at 15° C. or 40° C.

    ______________________________________                                        KOH test:    positive                                                         Aminopeptidase:                                                                            weak positive                                                    Oxidase:     weak positive                                                    Catalase:    positive                                                         NaCl tolerance:                                                                            obligate halophile, growth at 4% NaCl                                         no growth at 0% or 8% NaCl                                       Hydrolysis of Gelatin:                                                                     slow positive                                                    Hydrolysis of Starch:                                                                      positive                                                         Major ubiquinone:                                                                          Q9                                                               G + C:       64.1 (T.sub.M)                                                   ______________________________________                                    

Chemoorganotroph. Metabolically unreactive. No growth on simple sugarsor organic acids. Grows on complex substrates such as yeast extract andpeptones, and on some amino acids.

Strain 4E.1

An aerobic, Gram-negative, motile, rod-shaped bacterium, 1.7-5.2 μm×0.75μm.

Obligate alkaliphile, grows best between pH 8.2 and pH 10.9.

On alkaline-agar, (Medium A) forms smooth, opaque, beige or browncolored colonies, 2-4 mm in diameter. The colonies are circular in form,convex in elevation, with an entire margin.

In alkaline-broth (Medium A), growth (37° C.) is heavy and flocculentwith a sediment and surface pellicle.

Grows well between 20° C. and 37° C. Grows very slowly at 10° C. and notat all at 8° C. No growth at 40° C. or above.

    ______________________________________                                        KOH test:    positive                                                         Aminopeptidase:                                                                            positive                                                         Oxidase:     very weakly positive, can appear negative                        Catalase:    positive                                                         NaCl tolerance:                                                                            0% to 12%, may grow weakly at 15%                                             no growth at 20%                                                 Hydrolysis of Gelatin:                                                                     negative                                                         Hydrolysis of Starch:                                                                      negative                                                         ______________________________________                                    

Chemoorganotroph. Does not grow on simple sugars, except for ribose.Grows well on complex substrates such as yeast extract, and on organicacids (e.g., succinate, pyruvate, citrate, malonate, acetate andlactate), fatty acids (e.g., propionate, valerate and suberate), andamino acids (e.g., proline, serine, histidine and lysine).

Strain 5E.1

An aerobic, Gram-negative, rod-shaped bacterium, 3.0-5.3 μm×1.3 μm.

Obligate alkaliphile, grows best between pH 9 and pH 10.5.

On alkaline-agar, (Medium A) forms smooth, opaque, brown coloredcolonies, 3-4 mm in diameter. The colonies are fairly irregular in form,generally flat to slightly umbonate in elevation with a lobate margin.

In alkaline-broth (Medium A), growth (37° C.) is moderate to heavy,becoming flocculent with a sediment and surface pellicle.

Grows well between 20° C. and 40° C. Grows slowly at 10° C. No growth at45° C.

    ______________________________________                                        KOH test:        positive                                                     Aminopeptidase:  positive                                                     oxidase:         negative                                                     Catalase:        positive                                                     NaCl tolerance:  0% to 12%, may grow weakly                                                    at 15%                                                                        no growth at 20%                                             Hydrolysis of Gelatin:                                                                         positive                                                     Hydrolysis of Starch:                                                                          weakly positive                                              Major polar lipid components:                                                                  phosphatidylglycerol                                                          diphosphatidylglycerol                                                        phosphatidylglycerol phosphate                                                phosphatidylglycerol sulphate                                                 phosphatidylethanolamine                                                      glycolipid (α-naphthol positive)                       Major ubiquinone:                                                                              Q8                                                           Major fatty acids:                                                                             C16:0, C18:1                                                 ______________________________________                                    

Chemoorganotroph. Does not grow on simple sugars. Grows well on complexsubstrates such as yeast extract, organic acids (e.g., pyruvate,citrate, acetate and lactate), fatty acids (e.g., propionate, caprateand valerate) and amino acids (e.g., proline, alanine and lysine).

Strain 92LM.4

An aerobic, Gram-negative, rod-shaped bacterium, 2.0-3.5 μm×0.5-1.0 μm.

Obligate alkaliphile, no growth below pH 7.5.

On alkaline-agar, (Medium A) forms smooth, cream colored colonies,initially translucent but becoming opaque. The colonies develop fromcircular, entire to irregular, lobate in form, with a convex elevation.

In alkaline-broth (Medium A), growth (37° C.) is slow, slight,flocculent with a sediment but no surface pellicle.

Grows between 10° C. and 40° C., no growth at 8° C. or 45° C.

    ______________________________________                                        KOH test:       positive                                                      Aminopeptidase: negative                                                      Oxidase:        positive                                                      Catalase:       positive                                                      NaCl tolerance: 0% to 15%, growth at 15% is slow                                              no growth at 20%                                              Hydrolysis of Gelatin:                                                                        positive                                                      Hydrolysis of Starch:                                                                         weakly positive                                               Major ubiquinone:                                                                             Q9                                                            Major fatty acids:                                                                            C15:0 iso, C15:0 anteiso,                                                     C16:0, C17:0 iso                                              ______________________________________                                    

Chemoorganotroph. Grows on complex substrates such as yeast extract andpeptones, and a variety of sugars, organic acids and amino acids.

Strain wBn5

An aerobic, Gram-negative, small, rod-shaped bacterium, frequentlyforming short chains of cells.

Obligate alkaliphile, no growth below pH 8.

On alkaline-agar, (Medium A) forms smooth, circular, convex colonieswith an entire margin, about 1 mm in diameter. The colonies areinitially cream/beige, transparent becoming opaque, brown.

In alkaline-broth (Medium A), growth (37° C.) is initially evenly turbidwith a sediment but no surface pellicle becoming after 4 days flocculentwith formation of a pellicle.

Grows at 30° C. and 37° C. No growth at 40° C.

    ______________________________________                                        KOH test:    positive                                                         Aminopeptidase:                                                                            positive                                                         Oxidase:     positive                                                         Catalase:    positive                                                         NaCl tolerance:                                                                            obligate halophile. Growth at 4% NaCl                                         no growth at 0% or 8%                                            Hydrolysis of Gelatin:                                                                     slow positive                                                    Hydrolysis of Starch:                                                                      negative                                                         Major ubiquinone:                                                                          Q8, Q9                                                           G + C:       54.6 mol % (T.sub.M)                                             ______________________________________                                    

Chemoorganotroph. Grows on a range of complex substrates such as yeastextract and peptones, as well as sugars, organic acids, fatty acids andamino acids.

Cluster Definition by the Calculation of the Minimum Number ofDiscriminatory Tests, and the Construction of a Probability Matrix forthe Identification of Gram-Negative Alkaliphiles

One of the purposes of a numerical classification study is to use thephenetic data, which defines the clusters at a selected similaritylevel, for the assignment or identification of unknown strains. Theclassification test data cam be used to determine the minimum set oftests which are required to define the clusters at the 73% (S_(G))similarity level, and to identify those characters which are mostdiagnostic (predictive) for the individual clusters. In other words, theminimum number of tests required to assign an unknown organism to apre-determined cluster with a high degree of predictability.

From the minimum discriminatory tests, a probability matrix can beconstructed for the identification of unknown strains. The analysis isachieved by using a combination of the CHARSEP and DIACHAR (TAXPAK) andMCHOICE (not on TAXPAK but available by Data-Mail from the University ofLeicester, U.K.) programs. An evaluation of the identification matrix isprovided by using the MOSTTYP and OVERMAT programs. Practical examplesof the use of these programs for the probabilistic identification ofbacteria have been published by Williams, S. T., et al., (1983), J. Gen.Microbiol., 129, pp. 1815-1830; and Priest, F. G. and Alexander, B.,(1988), J. Gen. Microbiol., 134, pp. 3011-3018; ibid, (1990), 136, pp.367-376.

A "n×t" table was constructed from the test data using characters 6 to10 and 13 to 104 (Appendix C) scored in binary notation (positive=1,negative=0). This data matrix was supplemented with the following fourextra character states:

[105] Bright yellow colonies (character number 1, Appendix C)

[106] Translucent colonies (grown on Medium A, Appendix A)

[107] Lipase (lipolytic activity on olive oil (Medium M))

[108] Oxidase positive within 10 secs. (test 9, Appendix B)

The data matrix is first examined using the CHARSEP program whichcalculates separation indices and thus the diagnostic value of theindividual characters for discriminating between the clusters. Testswith a VSP index >25% (Sneath, P. H. A., (1979), Computers andGeosciences, 5, 349-357) are accepted, characters with a low diagnosticvalue (VSP<25%) were rejected. A preference is made for characters withthe highest VSP indices, provided that the criteria in the DIACHAR andMCHOICE programs are also met. In this example, 38 tests have a VSPindex >25%, and 9 of the 24 characters finally chosen have a VSPindex >50% (Table 11).

The data matrix is next re-examined by means of the DIACHAR program,which determines the most diagnostic character states of each of theclusters. The number of character states was set at 10. This resultallows the choice of mutually exclusive character states between theclusters. As many of these tests as possible are retained in the finalidentification matrix of minimum discriminatory tests; in this examplebetween 6 and 9 diagnostic characters per cluster. The remaining, unusedtests are also noted and may be applied as additional tests for theconfirmation of identification (Table 12).

The MCHOICE program ranks the tests in groups which can be displayed inthe form of a dendrogram using the MDEND subroutine. The groups identifytests with similar discriminatory value, thus allowing the rejection oftests which fail to make a significant discrimination as well asallowing choices to be made between tests of equal or very similardiagnostic value.

Table 13 shows the set of 24 tests which is the minimum number requiredto define the clusters and which can be used for the assignment ofunknown strains. In addition, Table 13 shows the identification matrixwhich consists of the percentage of positive characters which define theclusters on the basis of the 24 minimum discriminatory tests. This iscomputed by the IDMAT program.

                  TABLE 14                                                        ______________________________________                                        Separation Values of Characters used for the                                  Minimum Discriminatory Tests                                                  CHARACTER           VSP Index                                                 ______________________________________                                        [23]     N-acetylglucosamine                                                                          35.4                                                  [26]     Saccharose     44.8                                                  [27]     Maltose        41.4                                                  [32]     Lactate        51.6                                                  [41]     Propionate     60.9                                                  [43]     Valerate       63.4                                                  [44]     Citrate        45.1                                                  [45]     Histidine      38.0                                                  [47]     Glycogen       31.7                                                  [51]     3-hydroxybutyrate                                                                            66.1                                                  [52]     4-hydroxybenzoate                                                                            38.0                                                  [58]     Leucine arylamidase                                                                          36.6                                                  [59]     Valine arylamidase                                                                           50.5                                                  [64]     Phosphohydrolase                                                                             52.8                                                  [65]     α-galactosidase                                                                        33.9                                                  [85]     Ampicillin     36.8                                                  [92]     Fusidic Acid   68.7                                                  [93]     Methicillin    58.3                                                  [99]     Polymixin      62.8                                                  [102]    Vancomycin     48.3                                                  ______________________________________                                    

                  TABLE 15                                                        ______________________________________                                        Discriminatory Tests for Each of the Six Clusters (S.sub.G)                   Positive            Negative                                                  ______________________________________                                        Cluster 1: cream, circular, opaque, mucoid colonies.                          [58] Leucine arylamidase                                                                           [23]   N-acetylglucosamine (9%)                               (91%)           [27]   Maltose (9%)                                      [59] Valine arylamidase                                                                            [41]   Propionate (9%)                                        (91%)           [42]   Caprate                                           [64] Phosphohydrolase (91%)                                                                        [43]   Valerate (9%)                                     [99] Polymixin (89%) [44]   Citrate (9%)                                                           [45]   Histidine                                                              [47]   Glycogen (9%)                                                          [52]   4-hydroxybenzoate                                                      [65]   α-galactosidase                             Cluster 2: small, cream, translucent colonies.                                [21] Starch          [23]   N-acetylglucosamine                               [31] Acetate         [26]   Saccharose                                        [41] Propionate      [45]   Histidine                                         [43] Valerate        [50]   2-ketogluconate                                   [53] Preline         [52]   4-hydroxybenzoate                                 [107]                                                                              Lipase          [68]   α-glucosidase                               [108]                                                                              Oxidase (within 10 secs.)                                                                     [69]   β-glucosidase                                                     [92]   Fusidic Acid                                      Cluster 3: cream, opaque colonies.                                            [31] Acetate         [64]   Phosphohydrolase (3%)                             [32] Lactate         [65]   α-galactosidase (3%)                        [41] Propionate (94%)                                                                              [92]   Fusidic Acid (3%)                                 [43] Valerate (97%)  [96]   Tetracycline (3%)                                 [44] Citrate (94%)   [102]  Vancomycin                                        [51] 3-hydroxybutyrate                                                                             [104]  Bacitracin                                             (94%)                                                                    [53] Proline                                                                  [58] Leucine arylamidase                                                           (94%)                                                                    Cluster 4: beige to brown, opaque colonies.                                   [32] Lactate         [45]   Histidine                                         [33] Alanine         [85]   Ampicillin                                        [48] 3-hydroxybutyrate                                                                             [86]   Naladixic acid                                    [59] Valine arylamidase                                                                            [88]   Trimethoprim                                      [99] Polymixin       [89]   Penicillin G                                                           [93]   Methicillin                                       Cluster 5: bright yellow [105], opaque colonies.                              [64] Phosphohydrolase                                                                              [23]   N-acetylglucosamine                               [65] α-galactosidase                                                                         [32]   Lactate                                           [66] β-galactosidase                                                                          [33]   L-alanine                                         [85] Ampicillin      [34]   Mannitol                                          [92] Fusidic Acid    [41]   Propionate                                        [93] Methicillin     [42]   Caprate                                           [96] Tetracyclinee   [43]   Valerate                                          [102]                                                                              Vancomycin      [45]   Histidine                                         [104]                                                                              Bacitracin      [48]   3-hydroxybenzoate                                                      [51]   3-hydroxybutyrate                                                      [52]   4-hydroxybenzoate                                                      [99]   Polymixin                                         Cluster 6: cream, irregular, flat colonies.                                   [21] Starch          [17]   Pyruvate                                          [23] N-acetylglucosamine                                                                           [52]   4-hydroxybenzoate                                 [26] Saccharose      [58]   Leucine arylamidase                               [27] Maltose         [59]   Valine arylamidase                                [31] Acetate         [65]   α-galactosidase                             [33] Alanine         [99]   Polymixin                                         [44] Citrate                                                                  [47] Glycogen                                                                 [51] 3-hydroxybutyrate                                                        [89] Penicillin G                                                             [92] Fusidic Acid                                                             [93] Methicillin                                                              [96] Tetracyclinee                                                            [104]                                                                              Bacitracin                                                               ______________________________________                                         Note: The numbers in square brackets proceeding the character state refer     to the character states and unit tests in Appendices B and C. The             percentage in parenthesis refers to positive character states.           

                  TABLE 16                                                        ______________________________________                                        A Probability Matrix for the Identification of Alkaliphiles:                  Percentage Distribution of Positive Discriminatory Characters                 Which Define the Clusters of Gram-Negative Alkaliphilic                       Bacteria at the 73% Level (S.sub.G)                                                         CLUSTER                                                         TEST            1     2      3    4    5    6                                 ______________________________________                                        [23] N-acetylglucosamine                                                                          13    0    26   20   0    100                             [26] Saccharose     25    0    74   20   25   100                             [27] Maltose        25    0    68   60   50   100                             [32] Lactate        38    50   100  100  0    40                              [41] Propionate     0     100  91   60   0    80                              [43] Valerate       13    100  97   80   0    40                              [44] Citrate        13    50   94   20   50   100                             [45] Histidine      0     0    71   0    0    80                              [47] Glycogen       0     13   26   20   25   100                             [51] 3-hydroxybutyrate                                                                            13    25   94   100  0    100                             [52] 4-hydroxybenzoate                                                                            0     0    71   80   0    0                               [58] Leucine arylamidase                                                                          88    63   94   60   50   0                               [59] Valine arylamidase                                                                           88    25   65   100  25   0                               [64] Phosphohydrolase                                                                             88    13   3    20   75   40                              [65] α-galactosidase                                                                        0     0    3    20   75   0                               [85] Ampicillin     50    63   56   0    100  80                              [92] Fusidic Acid   25    0    3    20   100  100                             [93] Methicillin    50    13   50   0    100  100                             [99] Polymixin      88    50   81   100  0    0                               [102]                                                                              Vancomycin     13    13   3    20   100  75                              [105]                                                                              Yellow colony  0     0    0    0    100  0                               [106]                                                                              Translucent colony                                                                           0     100  3    0    0    0                               [107]                                                                              Lipase         0     100  21   0    0    0                               [108]                                                                              Oxidase (10 secs)                                                                            25    88   6    0    0    0                               ______________________________________                                    

Evaluation of the Discriminatory Tests and Assessment of the Reliabilityof Identification

The evaluation of the discriminatory tests has two aspects. Firstly, thevalidity of the tests can be analyzed using practical examples, whichcan be further evaluated using statistical theory, or the tests can bedirectly subjected to theoretical assessment using statistical methods.

ILLUSTRATION 1 A Practical Evaluation of the Discriminatory Tests

Many workers assess the accuracy of the discriminatory tests only byredetermining the character states of selected cluster representatives.This approach has been used here for the centrotype strains (see below).A far more stringent approach which is seldom applied, is to examine allthe strains which were used in the original numerical taxonomicanalysis. When subjected to cluster analysis using only the dataacquired from the derived set of minimum discriminatory tests, thereconstructed dendrogram can be compared with the original. Using onlythe 24 discriminatory tests previously described (Table 16), the data(two-state, binary form) for all 70 of the novel Gram-negativealkaliphilic bacteria were subjected to cluster analysis by the S_(G)/UPGMA method. The reconstructed dendrogram is reproduced in FIG. 3.This reconstructed dendrogram compares very favorably with the originaldendrogram (FIG. 1).

Although there has been some rearrangement of position of the clusters,their composition is largely unchanged and they are defined atapproximately the same similarity level as the original. Cluster 4however, has combined with Cluster 3, with a single strain moving toCluster 1. This further serves to emphasize the difficulty of definingCluster 4 on phenetic data alone. It has been stressed several timesthat supplementary chemotaxonomic data are required to make the properdistinction between Cluster 3 and Cluster 4.

In both the original dendrogram and the reconstruction (FIG. 3), Cluster3 appears to comprise several sub-clusters above the 73% similaritylevel. The fine structure of cluster 3 is also supported by thechemotaxonomic data (see above).

ILLUSTRATION 2 A Theoretical Evaluation of the Discriminatory Tests

An assessment of cluster overlap is achieved using the OVERMAT program.This program examines the matrix constructed from the percentagepositive values for the selected character states against a criticaloverlap value by considering the clusters defined by the coordinates ofthe centroid and the cluster radius (twice root mean square of thedistances of the strains from the centroid). If there is significantoverlap between the clusters, unknown strains may not identify withsufficient confidence to any one of them (Sneath, P. H. A. and Sokal, R.R., supra, p. 394-400). At a chosen critical overlap value of 2.5%(which is a more stringent condition than is used by most workers: seePriest, F. G. and Alexander, B., (1988), supra; and Williams, S. T. etal., (1983), supra) there was no significant overlap between theclusters (95% confidence level) except between Cluster 3 and Cluster 4where the actual overlap was calculated to be 4%. However,chemotaxonomic data (see above) was not taken into account whenconstructing the identification matrix. On the basis of quinoneanalyses, strains from Cluster 3 can be distinguished from the strainsof Cluster 4.

ILLUSTRATION 3 A Theoretical Assessment of the Reliability ofIdentification

The hypothetical median organism (HMO) is another estimate of the"average" organism in a cluster (Sneath, P. H. A. and Sokal, R. R.,supra, pp. 194 et seq.). A HMO is not a real strain but a hypotheticalorganism possessing the most common state for each character. TheMOSTTYP program calculates HMO's for each cluster in the identificationmatrix and then attempts to identify them. In other words, MOSTTYP is aprogram to evaluate an identification matrix by calculatingidentification scores of the most typical strains against the clusters.A good identification matrix should give a high probability of a HMObeing reassigned to its own cluster. The results of this analysis werevery satisfactory (Table 17), especially since MOSTTYP was programed toconsider only the first 20 diagnostic tests of the identification matrix(Table 16), i.e. excluding tests 105-108. Each HMO was reassigned to itsoriginal cluster with Willcox probabilities of 0.998-1.000 (Willcox, W.R. et al., (1973) J. Gen. Microbiol., 77, 317-330). The TaxonomicDistances were all low and the standard errors of the Taxonomic Distancewere all negative, indicating that the HMO's were all closer to thecentroid of the cluster than the average for the cluster (Table 17).

                  TABLE 17                                                        ______________________________________                                        Identification Scores for the Hypothetical Median Organism                    of each cluster provided by the MOSTTYP Program                                      Identification Score                                                            Willcox    Taxonomic  Standard Error of                              CLUSTER  Probability                                                                              Distance   Taxonomic Distance                             ______________________________________                                        1        0.999      0.194      -2.742                                         2        0.999      0.236      -2.214                                         3        1.000      0.231      -2.115                                         4        0.998      0.195      -2.998                                         5        1.000      0.217      -1.839                                         6        1.000      0.182      -2.502                                         ______________________________________                                    

ILLUSTRATION 4 A Practical Evaluation of Identification Score

Identification of strains using the minimum set of discriminatory testsis achieved using the MATIDEN program in TAXPAK. The program comparespresence-absence data for an unknown strain against each cluster in turnin an identification matrix of percentage positive characters.Identification coefficients are computed, namely Willcox probability,Taxonomic Distance and the Standard Error of the Taxonomic Distance. Theresults are displayed, showing the identification scores to the bestcluster and to the two next best alternative clusters. Additionally, theatypical results ("characters against") are recorded. In an analysisusing data from real strains, the centrotypes were reassigned to theiroriginal clusters with Willcox probabilities of 0.9996-1.000 (Table 18).The Taxonomic Distances were low. The Standard Errors of the TaxonomicDistance were all negative indicating that the centrotypes were closerto the centroid of the cluster than the average for the cluster.

                  TABLE 18                                                        ______________________________________                                        Identification Scores for the Centrotype Organisms of                         Each Cluster Provided by the MATIDEN Program                                               Identification Score                                                           Assigned         Taxonomic                                      Clus-         to       Willcox Distance                                                                              Standard                               ter  Strain   Cluster  Probability                                                                           (D)     Error of D                             ______________________________________                                        1    2E.1     1        1.000   0.309   -0.283                                 2    45E.3.sup.CT                                                                           2        1.000   0.226   -1.749                                 3    28N.1.sup.CT                                                                           3        1.000   0.305   -0.622                                 4    wB4.sup.CT                                                                             4         0.9996 0.265   -1.092                                 5    17N.1.sup.CT                                                                           5         0.9999 0.255   -0.478                                 6    64N.4.sup.CT                                                                           6        1.000   0.211   -1.126                                 ______________________________________                                    

ILLUSTRATION 5 Identification of Unknown Isolates

The identification matrix was assessed for the ability to assign unknownGram-negative alkaliphiles to the clusters defined herein. The criteriafor a successful identification were:

(a) bacteria isolated from a habitat similar to, but geographicallyseparate from, the East African soda lakes;

(b) a Willcox probability greater than 0.95 and low values for TaxonomicDistance and its standard error (<3);

(c) an identification score to the best cluster significantly betterthan those against the two next best alternatives;

(d) "characters against" the best cluster should be zero or few innumber.

Unknown microorganisms may be examined using the minimum tests listed inTable 16. The character states are determined and identification scoresobtained using the MATIDEN program. This program compares the characterstates of the unknown with the identification matrix determined for allof the predetermined clusters, computes the best match and assigns theunknown to the most appropriate cluster.

A Willcox probability is calculated to determine the acceptability ofidentification. Willcox probabilities of 0.85 and 0.95 have beenaccepted as criteria for a successful identification (Williams, S. T.,et al. (1983), supra; Priest, F. G. and Alexander, B., (1988), supra).The Taxonomic Distance of the unknown from the cluster centroid iscalculated and may be compared to the radius of the cluster. TheStandard Error of the Taxonomic Distance should be less than the uppervalue of +3.0 suggested by Sneath, P. H. A. ((1979), pp. 195-213).Moreover, physical characteristics, additional biochemical data andchemotaxomomic markers may be used to further confirm the identity ofthe unknown in a particular cluster.

The results provided by these five illustrations, together with thestatistical data provided by the numerical taxonomic analysis and thechemotaxonomic data, indicate a robust classification which identifies 6major groups of new, Gram-negative, alkaliphilic bacteria.

Production and Application of Alkali-Tolerant Enzymes

The alkaliphilic microorganisms of the present invention produce avariety of alkali-tolerant enzymes. Examples of enzyme activitiespresent in representative strains of the Gram-negative bacteria of thepresent invention may be found in Appendices D and E. These enzymes arecapable of performing their functions at an extremely high pH, makingthem uniquely suited for their application in a variety of processesrequiring such enzymatic activity in high pH environments or reactionconditions.

Examples of the various applications for alkali-tolerant enzymes are indetergent compositions, leather tanning, food treatment, waste treatmentand in the textile industry. These enzymes may also be used forbiotransformations, especially in the preparation of pure enantiomers.

The alkaliphilic bacteria of the present invention may easily bescreened for the production of alkali-tolerant lipases, proteases andstarch-degrading enzymes, inter alia, using the methods describedherein.

The broth in which alkaliphilic bacteria are cultured typically containsone or more types of enzymatic activity. The broth containing the enzymeor enzymes may be used directly in the desired process after the removalof the bacteria therefrom by means of centrifugation or filtration, forexample.

If desired, the culture filtrate may be concentrated by freeze drying,before or after dialysis, or by ultrafiltration. The enzymes may also berecovered by precipitation and filtration. Alternatively, the enzyme orenzymes contained in the broth may be isolated and purified bychromatographic means or by gel electrophoresis, for example, beforebeing applied to the desired process. The exact methods used to treatthe culture filtrate and/or to extract and/or purify the alkali-tolerantenzymes is not critical to the present invention, and may be determinedby one skilled in the art.

The genes encoding alkali-tolerant enzymes of interest may be cloned andexpressed in organisms capable of expressing the desired enzyme in apure or easily recoverable form.

The following examples are provided to illustrate methods for theidentification of Gram-negative alkaliphilic bacteria of the presentinvention, as well as method of screening these alkaliphilic bacteriafor the presence of various alkali-tolerant enzymes and methods for thesubsequent production and application of these enzymes in industrialprocesses. These examples are not to be construed so as to limit thescope of the present invention.

EXAMPLE 1 Identification of Unknown Isolates

Six strains of Gram-negative alkaliphilic bacteria were isolated fromMono lake, a hypersaline, alkaline lake situated in California, U.S.A.(Javor, B., in Hypersaline Environments, Springer Verlag, Berlin andHeidelberg (1988), pp. 303-305). The strains were isolated from samplesof partially submerged soda-encrusted wood, tufa and soda-soil collectedfrom the environs of Mono lake (California, U.S.A.) in May, 1990 byenrichment culture at 37° C. in Medium A (Appendix A). The six strainsare described in Table 19. The strains were examined using 21 of the 24minimum tests listed in Table 16. The character states were determinedand identification scores obtained using the MATIDEN program. Theresults are outlined in Table 20.

                  TABLE 19                                                        ______________________________________                                        Alkaliphilic Strains from Mono Lake                                                   Colony               Cell                                             Strain                                                                              Sample  Color   Form   Elevation                                                                             Margin                                                                              Shape                              ______________________________________                                        ML005 tufa    beige   circular                                                                             convex  entire                                                                              rod                                ML104 wood    pink/   circular                                                                             convex  entire                                                                              rod                                              beige                                                           ML201 wood    yellow  circular                                                                             convex  entire                                                                              rod                                ML203 wood    pink/   circular                                                                             convex  entire                                                                              rod                                              beige                                                           ML206 wood    yellow  circular                                                                             convex  entire                                                                              short                                                                         rod                                ML301 soil    beige   circular                                                                             convex  entire                                                                              rod                                ______________________________________                                    

                  TABLE 20                                                        ______________________________________                                        Examine of the Output from the MATIDEN Program to Identify                    Six Unknown Strains against the Identification Matrix                         ______________________________________                                        A. Reference Number of unknown is ML005                                                          Percent in:                                                                Value in           Next Best                                  Character       unknown  Best Taxon                                                                              Taxon                                      ______________________________________                                        [23] N-acetylglucosamine                                                                          +        26      20                                       [26] Saccharose     +        74      20                                       [27] Maltose        +        68      60                                       [32] Lactate        +        99      99                                       [41] Propionate     +        91      60                                       [43] Valerate       +        97      80                                       [44] Citrate        +        94      20                                       [45] Histidine      +        71      1                                        [47] Glycogen       +        26      20                                       [51] 3-Hydroxybutyrate                                                                            +        94      99                                       [52] 4-Hydroxybenzoate                                                                            -        71      80                                       [58] Leucine arylamidase                                                                          +        94      60                                       [59] Valine arylamidase                                                                           +        65      99                                       [64] Phosphohydrolase                                                                             -        3       20                                       [65] α-Galactosidase                                                                        n.t.     3       20                                       [85] Ampicillin     -        56      1                                        [92] Fusidic Acid   -        3       20                                       [93] Methicillin    -        50      1                                        [99] Polymix        +        81      99                                       [102]                                                                              Vancomycin     -        3       20                                       [105]                                                                              Yellow colony  -        1       1                                        [106]                                                                              Translucent colony                                                                           n.t.     3       1                                        [107]                                                                              Lipase         n.t.     21      1                                        [108]                                                                              Oxidase (10 sec.)                                                                            +        6       1                                        ______________________________________                                        Isolate ML005 best identification is Cluster 3. Scores                        for coefficients: 1 (Willcox probability), 2 (Taxonomic Dis-                  tance), 3 (Standard Error of Taxonomic Distance).                                       1               2      3                                            ______________________________________                                        Cluster 3 0.9999          0.407  1.475                                        Cluster 4 0.8266 × 10.sup.-4                                                                      0.526  4.590                                        Cluster 2 0.4086 × 10.sup.-7                                                                      0.584  6.296                                        ______________________________________                                                      Cluster 3                                                       Characters against                                                                           % in Taxon      Value in unknown                               ______________________________________                                        [108]                                                                              Oxidase (10 sec.)                                                                           6             +                                            ______________________________________                                        Additional characters that                                                                   Cluster 3        Cluster 4                                     assist in separating                                                                         %         from   %                                             ______________________________________                                        [106]                                                                              Translucent colony                                                                          3              99                                          [107]                                                                              Lipase        21             99                                          ______________________________________                                        B. Reference Number of unknown is ML104                                                          Percent in:                                                                Value in           Next Best                                  Character       unknown  Best Taxon                                                                              Taxon                                      ______________________________________                                        [23] N-acetylglucosamine                                                                          -        13      1                                        [26] Saccharose     -        25      1                                        [27] Maltose        -        25      1                                        [32] Lactate        -        38      50                                       [41] Propionate     -        1       99                                       [43] Valerate       -        13      99                                       [44] Citrate        -        13      50                                       [45] Histidine      -        1       1                                        [47] Glycogen       -        1       13                                       [51] 3-Hydroxybutyrate                                                                            -        13      25                                       [52] 4-Hydroxybenzoate                                                                            -        1       1                                        [58] Leucine arylamidase                                                                          +        88      63                                       [59] Valine arylamidase                                                                           +        88      25                                       [64] Phosphohydrolase                                                                             +        88      13                                       [65] α-Galactosidase                                                                        n.t.     1       1                                        [85] Ampicillin     -        50      63                                       [92] Fusidic Acid   -        25      1                                        [93] Methicillin    -        50      13                                       [99] Polymix        +        88      50                                       [102]                                                                              Vancomycin     -        13      13                                       [105]                                                                              Yellow colony  -        1       1                                        [106]                                                                              Translucent colony                                                                           n.t.     1       99                                       [107]                                                                              Lipase         n.t.     1       99                                       [108]                                                                              Oxidase (10 sec.)                                                                            +        25      88                                       ______________________________________                                        Isolate ML104 best identification is Cluster 1. Scores                        for coefficients: 1 (Willcox probability), 2 (Taxonomic Dis-                  tance), 3 (Standard Error of Taxonomic Distance).                                       1               2      3                                            ______________________________________                                        Cluster 1 0.9999          0.271  -1.108                                       Cluster 2 0.825 × 10.sup.-5                                                                       0.473  3.797                                        Cluster 4 0.407 × 10.sup.-7                                                                       0.534  4.767                                        ______________________________________                                                      Cluster 1                                                       Characters against                                                                           % in Taxon      Value in unknown                               ______________________________________                                        (none)                                                                        ______________________________________                                        Additional characters that                                                                   Cluster 1        Cluster 2                                     assist in separating                                                                         %         from   %                                             ______________________________________                                        [106]                                                                              Translucent colony                                                                          1              99                                          [107]                                                                              Lipase        1              99                                          ______________________________________                                                       Cluster 1        Cluster 4                                                    %         from   %                                             ______________________________________                                        (none)                                                                        ______________________________________                                        C. Reference Number of unknown is ML201                                                          Percent in:                                                                Value in           Next Best                                  Character       unknown  Best Taxon                                                                              Taxon                                      ______________________________________                                        [23] N-acetylglucosamine                                                                          -        1       13                                       [26] Saccharose     -        25      25                                       [27] Maltose        -        50      25                                       [32] Lactate        -        1       38                                       [41] Propionate     -        1       1                                        [43] Valerate       -        1       13                                       [44] Citrate        -        50      13                                       [45] Histidine      -        1       1                                        [47] Glycogen       -        25      1                                        [51] 3-Hydroxybutyrate                                                                            -        1       13                                       [52] 4-Hydroxybenzoate                                                                            -        1       1                                        [58] Leucine arylamidase                                                                          +        50      88                                       [59] Valine arylamidase                                                                           +        25      88                                       [64] Phosphohydrolase                                                                             +        75      88                                       [65] α-Galactosidase                                                                        n.t.     75      1                                        [85] Ampicillin     +        99      50                                       [92] Fusidic Acid   +        99      25                                       [93] Methicillin    +        99      50                                       [99] Polymix        -        1       88                                       [102]                                                                              Vancomycin     +        99      13                                       [105]                                                                              Yellow colony  +        99      1                                        [106]                                                                              Translucent colony                                                                           n.t.     1       1                                        [107]                                                                              Lipase         n.t.     1       1                                        [108]                                                                              Oxidase (10 sec.)                                                                            -        1       25                                       ______________________________________                                        Isolate ML201 best identification is Cluster 5. Scores                        for coefficients: 1 (Willcox probability), 2 (Taxonomic Dis-                  tance), 3 (Standard Error of Taxonomic Distance).                                       1               2      3                                            ______________________________________                                        Cluster 5 0.9999          0.267  -0.176                                       Cluster 1 0.835 × 10.sup.-4                                                                       0.437  2.435                                        Cluster 2 0.177 × 10.sup.-11                                                                      0.641  7.593                                        ______________________________________                                                      Cluster 5                                                       Characters against                                                                           % in Taxon      Value in unknown                               ______________________________________                                        (none)                                                                        ______________________________________                                        Additional characters that                                                                   Cluster 5        Cluster 1                                     assist in separating                                                                         %         from   %                                             ______________________________________                                        [65] α-Galactosidase                                                                       75              1                                          ______________________________________                                                     Cluster 5      Cluster 2                                                      %       from   %                                                 ______________________________________                                        [65] α-Galactosidase                                                                       75              1                                          [106]                                                                              Translucent colony                                                                           1             99                                          [107]                                                                              Lipase         1             99                                          ______________________________________                                        D. Reference Number of unknown is ML203                                                          Percent in:                                                                Value in           Next Best                                  Character       unknown  Best Taxon                                                                              Taxon                                      ______________________________________                                        [23] N-acetylglucosamine                                                                          +        26      20                                       [26] Saccharose     +        74      20                                       [27] Maltose        +        68      60                                       [32] Lactate        +        99      99                                       [41] Propionate     +        91      60                                       [43] Valerate       +        97      80                                       [44] Citrate        +        94      20                                       [45] Histidine      +        71      1                                        [47] Glycogen       +        26      20                                       [51] 3-Hydroxybutyrate                                                                            +        94      99                                       [52] 4-Hydroxybenzoate                                                                            +        71      80                                       [58] Leucine arylamidase                                                                          +        94      60                                       [59] Valine arylamidase                                                                           +        65      99                                       [64] Phosphohydrolase                                                                             -        3       20                                       [65] α-Galactosidase                                                                        n.t.     3       20                                       [85] Ampicillin     -        56      1                                        [92] Fusidic Acid   -        3       20                                       [93] Methicillin    -        50      1                                        [99] Polymix        +        81      99                                       [102]                                                                              Vancomycin     +        3       20                                       [105]                                                                              Yellow colony  -        1       1                                        [106]                                                                              Translucent colony                                                                           n.t.     3       1                                        [107]                                                                              Lipase         n.t.     21      1                                        [108]                                                                              Oxidase (10 sec.)                                                                            +        6       1                                        ______________________________________                                        Isolate ML203 best identification is Cluster 3. Scores                        for coefficients: 1 (Willcox probability), 2 (Taxonomic Dis-                  tance), 3 (Standard Error of Taxonomic Distance).                                       1               2      3                                            ______________________________________                                        Cluster 3 0.9989          0.437  2.076                                        Cluster 4 0.1090 × 10.sup.-2                                                                      0.526  4.590                                        Cluster 2 0.8137 × 10.sup.-9                                                                      0.650  7.793                                        ______________________________________                                                      Cluster 3                                                       Characters against                                                                           % in Taxon      Value in unknown                               ______________________________________                                        [102]                                                                              Vancomycin    3             +                                            [108]                                                                              Oxidase (10 sec.)                                                                           6             +                                            ______________________________________                                        Additional characters that                                                                   Cluster 3        Cluster 4                                     assist in separating                                                                         %         from   %                                             ______________________________________                                        (none)                                                                        ______________________________________                                        E. Reference Number of unknown is ML206                                                          Percent in:                                                                Value in           Next Best                                  Character       unknown  Best Taxon                                                                              Taxon                                      ______________________________________                                        [23] N-acetylglucosamine                                                                          -        1       13                                       [26] Saccharose     +        25      25                                       [27] Maltose        -        50      25                                       [32] Lactate        -        1       38                                       [41] Propionate     -        1       1                                        [43] Valerate       -        1       13                                       [44] Citrate        -        50      13                                       [45] Histidine      -        1       1                                        [47] Glycogen       +        25      1                                        [51] 3-Hydroxybutyrate                                                                            -        1       13                                       [52] 4-Hydroxybenzoate                                                                            -        1       1                                        [58] Leucine arylamidase                                                                          +        50      88                                       [59] Valine arylamidase                                                                           +        25      88                                       [64] Phosphohydrolase                                                                             +        75      88                                       [65] α-Galactosidase                                                                        n.t.     75      1                                        [85] Ampicillin     +        99      50                                       [92] Fusidic Acid   +        99      25                                       [93] Methicillin    +        99      50                                       [99] Polymix        -        1       88                                       [102]                                                                              Vancomycin     +        99      13                                       [105]                                                                              Yellow colony  +        99      1                                        [106]                                                                              Translucent colony                                                                           n.t.     1       1                                        [107]                                                                              Lipase         n.t.     1       1                                        [108]                                                                              Oxidase (10 sec.)                                                                            -        1       25                                       ______________________________________                                        Isolate ML206 best identification is Cluster 5. Scores                        for coefficients: 1 (Willcox probability), 2 (Taxonomic Dis-                  tance), 3 (Standard Error of Taxonomic Distance).                                       1               2      3                                            ______________________________________                                        Cluster 5 0.9999          0.345  1.766                                        Cluster 1 0.2530 × 10.sup.-5                                                                      0.512  4.013                                        Cluster 6 0.2531 × 10.sup.-13                                                                     0.652  10.883                                       ______________________________________                                                      Cluster 5                                                       Characters against                                                                           % in Taxon      Value in unknown                               ______________________________________                                        (none)                                                                        ______________________________________                                        Additional characters that                                                                   Cluster 5        Cluster 1                                     assist in separating                                                                         %         from   %                                             ______________________________________                                        [65] α-Galactosidase                                                                       75             1                                           ______________________________________                                        F. Reference Number of unknown is ML301                                                          Percent in:                                                                Value in           Next Best                                  Character       unknown  Best Taxon                                                                              Taxon                                      ______________________________________                                        [23] N-acetylglucosamine                                                                          -        26      1                                        [26] Saccharose     +        74      1                                        [27] Maltose        +        68      1                                        [32] Lactate        +        99      50                                       [41] Propionate     +        91      99                                       [43] Valerate       +        97      99                                       [44] Citrate        +        94      50                                       [45] Histidine      +        71      1                                        [47] Glycogen       +        26      13                                       [51] 3-Hydroxybutyrate                                                                            +        94      25                                       [52] 4-Hydroxybenzoate                                                                            -        71      1                                        [58] Leucine arylamidase                                                                          +        94      63                                       [59] Valine arylamidase                                                                           +        65      25                                       [64] Phosphohydrolase                                                                             -        3       13                                       [65] α-Galactosidase                                                                        n.t.     3       1                                        [85] Ampicillin     +        56      63                                       [92] Fusidic Acid   -        3       1                                        [93] Methicillin    +        50      13                                       [99] Polymix        +        81      50                                       [102]                                                                              Vancomycin     -        3       13                                       [105]                                                                              Yellow colony  -        1       1                                        [106]                                                                              Translucent colony                                                                           n.t.     3       99                                       [107]                                                                              Lipase         n.t.     21      99                                       [108]                                                                              Oxidase (10 sec.)                                                                            +        6       88                                       ______________________________________                                        Isolate ML301 best identification is Cluster 3. Scores                        for coefficients: 1 (Willcox probability), 2 (Taxonomic Dis-                  tance), 3 (Standard Error of Taxonomic Distance).                                       1               2      3                                            ______________________________________                                        Cluster 3 1.000           0.429  1.918                                        Cluster 2 0.2841 × 10.sup.-6                                                                      0.603  6.731                                        Cluster 4 0.9313 × 10.sup.-8                                                                      0.623  6.704                                        ______________________________________                                                      Cluster 3                                                       Characters against                                                                           % in Taxon      Value in unknown                               ______________________________________                                        [108]                                                                              Oxidase (10 sec.)                                                                           6             +                                            ______________________________________                                        Additional characters that                                                                   Cluster 3        Cluster 4                                     assist in separating                                                                         %         from   %                                             ______________________________________                                        [106]                                                                              Translucent colony                                                                          3              99                                          [107]                                                                              Lipase        21             99                                          ______________________________________                                         n.t. = not tested                                                        

EXAMPLE 2 Production of Proteolytic Enzymes

Two alkaliphilic strains (1E.1 and 9B.1) were tested for the productionof proteolytic enzyme(s) in 7 different media poised at an alkaline pH.The experiments were carried out in liter shake flasks with a baffle,each of the flasks contained 400 ml of the nutrient media R to X(Appendix A). The flasks were placed in an orbital incubator rotating atrevolutions per minute at a constant temperature of 37° C. Samples ofculture media were removed from the flasks at intervals of 1, 2, 3, 4,5, 6 and 8 days for the determination of enzyme content which isexpressed in Alkaline Delft Units (ADU--as described in British PatentSpecification 1,353,317).

Table 21 presents the maximum enzyme yields and the pH of thecultivation medium at the moment at which the measurement of enzymelevels were made.

                  TABLE 21                                                        ______________________________________                                        Production of Proteolytic Enzymes                                                    STRAIN 1E.1.sup.CT                                                                          STRAIN 9B.1                                                                pH of             pH of                                     MEDIUM   ADU/ml   MEDIUM     ADU/ml MEDIUM                                    ______________________________________                                        R        100      8.2        14     9.7                                       S        140      8.5        49     9.1                                       T        111      8.7        6      9.1                                       U         6       9.7        4      9.7                                       V         51      9.5        7      9.6                                       W         94      9.2        7      9.3                                       X        100      9.6        28     9.6                                       ______________________________________                                    

The results of the test clearly indicate the presence of proteolyticenzymes, produced by the alkaliphilic bacteria of the present invention,in the culture broth.

EXAMPLE 3 Wash Performance Test Using Proteolytic Enzymes

Enzyme preparations from the alkaliphilic bacteria were tested in aspecially developed mini-wash test using cotton swatches (2.5×2.5 cm)soiled with milk, blood and ink (obtained from EMPA, St. Gallen,Switzerland, and designated EMPA 116). Prior to the wash test theswatches were pre-treated with a solution containing an anionicsurfactant, sodium perborate and a bleach activator (TAED) at ambienttemperature for 15 minutes. After this treatment the test swatches wererinsed in running demineralized water for 10 minutes and air-dried. Thistreatment results in the fixation of the soil, making its removal moredifficult.

The washing tests were performed in 100 ml Erlenmeyer flasks providedwith a baffle and containing 30 ml of a defined detergent compositionplus 300 ADU protease to be tested. In each flask were placed twopre-treated EMPA 116 test swatches. The flasks were placed in areciprocal shaking water bath (2 cm stroke) and agitated at 320revolutions per minute. The tests were carried out at 40° C. for 30minutes. After washing, the swatches were rinsed in runningdemineralized water for 10 minutes and air-dried. The reflectance of thetest swatches was measured at 680 nm with a Photovolt photometer (Model577) equipped with a green filter.

The wash performance of the supernatant fraction of cultures of variousalkaliphilic bacteria in European powder detergents was determinedaccording to the method specified above. The supernatant fractions weresubjected to various treatments so as to produce enzyme-containingpreparations.

100 ml Erlenmeyer flasks were charged with powder detergent IECdissolved in standard tap water of 15° German Hardness so as to give afinal concentration of 4 g per liter.

The composition of the powder detergent IEC was as follows:

    ______________________________________                                        Component                wt %                                                 ______________________________________                                        Linear sodium alkyl benzene sulphonate                                                                 6.4                                                  (mean chain length of alkane chain (C11.5))                                   Ethoxylated tallow alcohol (14EO)                                                                      2.3                                                  Sodium soap              2.8                                                  Sodium tripolyphosphate (STPP)                                                                         35.0                                                 Sodium silicate          6.0                                                  Magnesium silicate       1.5                                                  Carboxy methyl cellulose 1.0                                                  Sodium sulphate          16.8                                                 Sodium perborate tetrahydrate                                                                          18.5                                                 TAED                     1.5                                                  Miscellaneous + water    up to 100                                            ______________________________________                                         Standard tap water is composed of CaCl.sub.2.2H.sub.2 O, 0.291 g/l;           MgCl.6H.sub.2 O, 0.140 g/l and NaHCO.sub.3, 0.210 g/l dissolved in            demineralized water.                                                     

To each flask, two EMPA 116 swatches were added and sufficientenzyme-containing preparations to give a final activity of 300 ADU. Thefinal volume of the sud was 30 ml. By way of comparison, one flaskcontained no enzyme preparation, which was replaced with water. Thetrial was repeated either two or three times. The results are shown inTable 22.

                  TABLE 22                                                        ______________________________________                                        Application Washing Trials                                                    Performance of Proteolytic Enzyme-Containing Preparations                     in a Washing Formulation.                                                     Preparation                                                                   from                                                                          Strain   Average Remission of EMPA 116 Test Swatches                          ______________________________________                                        Untreated Culture Supernatant                                                          Trial 1       Trial 2 Trial 3                                        None     11.4          11.8    13.0                                           (control)                                                                     1E.1.sup.CT                                                                            29.2          22.2    24.7                                           9B.1     23.7          23.9    24.2                                           17N.1CT                11.8    18.4                                           24B.1                  17.3    16.3                                           Freeze Dried Supernatant Fraction                                                      Trial 1       Trial 2 Trial 3                                        None     10.4          11.8    13.0                                           (control)                                                                     1E.1.sup.CT                                                                            15.1          28.9    30.0                                           9B.1     21.7          14.9    17.4                                           17N.1.sup.CT           13.7    17.9                                           24B.1                  17.8    17.3                                           Dialyzed Supernatant Fractions                                                         Trial 1       Trial 2 Trial 3                                        None     11.4          11.8    13.0                                           (control)                                                                     1E.1.sup.CT                                                                            26.4          22.7    26.3                                           9B.1     18.7          16.7    17.0                                           17N.1.sup.CT           12.0    12.6                                           24B.1                  12.6    12.4                                           Ultrafiltration Concentrate of Supernatant Fractions                                   Trial 1       Trial 2                                                None     10.4          11.4                                                   (control)                                                                     1E.1.sup.CT                                                                            14.6          26.0                                                   9B.1     15.5          16.1                                                   Acetone Precipitates of Supernatant Fractions                                          Trial 1       Trial 2                                                None     10.4          11.4                                                   (control)                                                                     1E.1.sup.CT                                                                            13.4          23.4                                                   9B.1     12.6          14.7                                                   ______________________________________                                    

The results of the trials demonstrate the efficacy of the proteolyticenzymes produced by the strains of the present invention, provided invarious forms, in detergent formulations and the improved washingperformance obtained.

EXAMPLE 4 Production of Starch Degrading Enzymes

Strain 1E.1^(CT) was tested for the production of starch degradingenzymes on a starch containing medium poised at an alkaline pH.

500 ml Erlenmeyer flasks were charged with 100 ml of alkaline medium(Medium Y, Appendix A) containing 2% soluble starch. The flasks wereinoculated (5%) with cells of strain 1E.1^(CT) grown for 24 hours onMedium A (37° C.). As controls, similar flasks of alkaline medium notcontaining starch were also inoculated.

The flasks were placed in an orbital shaking incubator rotating at 280revolutions per minute, at a constant temperature of 37° C. for 24hours. The fluid containing the enzyme activity was separated from thecells by centrifugation for 10 minutes at 4000 r.p.m.

The enzyme activity of the supernatant was determined using the reducingsugar assay of Nelson and Somogyi (Methods in Microbiology, volume 5B,pp. 300-301; (eds. J. R. Norris and D. W. Ribbons), Academic Press,London, 1971).

DETERMINATION OF STARCH DEGRADING ENZYME ACTIVITY BY THE REDUCING SUGARASSAY Solutions

Reagent 1

144 g Na₂ SO₄ is dissolved by gentle warming in 500 ml demineralizedwater. 12 g potassium sodium tartrate tetrahydrate, 24 g Na₂ CO₃ and 16g NaHCO₃ are added to the solutions. The total volume of the solution isbrought to 800 ml by the addition of demineralized water.

Reagent 2

36 g Na₂ SO₄ is dissolved by gentle warming in 100 ml demineralizedwater and 4 g CuSO₄.5H₂ O is added to the warmed solution. The totalvolume of the solution is brought to 200 ml by the addition ofdemineralized water.

Directly before use, Reagents 1 and 2 are mixed in the ratio of 4:1(Reagent 1:Reagent 2).

Reagent 3

25 g ammonium molybdate tetrahydrate is dissolved in 450 mldemineralized water and 21 ml concentrated sulphuric acid is added withthorough mixing. 3 g Na₂ HAsO₄.7H₂ O are dissolved in 25 mldemineralized water and this solution is added to the molybdatesolution. The total solution is warmed for 48 hours at 37° C. and anyprecipitate is filtered off.

Standard

100 mg glucose is dissolved in demineralized water and the total volumeis brought to 100 ml. Before use, the solution is diluted 10 fold withdemineralized water.

Substrate

0.25% soluble starch (Merck, product number 1257) dissolved in 0.1M Na₂CO₃ --NaHCO₃ buffer, pH 10.1.

Assay

0.9 ml starch substrate solution, pH 10.1 is placed in a test-tube. Thetest-tube is placed in a water bath at 25° C. and allowed toequilibrate. The enzyme reaction is started by adding 0.1 ml of theenzyme-containing culture supernatant. The reaction is allowed toproceed for 30 minutes. The reaction is stopped by adding 1 ml ofReagent 1/2 and heating for 10 minutes at 100° C. The mixture is cooledon ice for 5 minutes and then 0.5 ml of Reagent 3 is added and the bluecolor is allowed to develop during 30 minutes at room temperature. Themixture is diluted by adding 1.0 ml demineralized water and theextinction is measured at 500 nm in a spectrophotometer. The reducingsugars are measured as glucose equivalents from a standard curve.

One unit of starch degrading enzyme activity is defined as 1 μg ofreducing sugars measured as glucose released per milliliter per minuteat pH 10.1 and 25° C.

The number of starch degrading enzyme units formed is shown in Table 23.

                  TABLE 23                                                        ______________________________________                                        Production of Starch Degrading Enzymes by Strain 1E.1                                  OPTICAL DENSITY                                                                              FINAL     ENZYME                                      MEDIUM   at 550 nm      pH        units per liter                             ______________________________________                                        plus starch                                                                            2.25           9.4       1150                                        no starch                                                                              0.75           10.3       660                                        ______________________________________                                    

The results of the test clearly indicate the presence of starchdegrading enzymes, produced by the alkaliphilic bacterial strain of thepresent invention, in the culture broth.

EXAMPLE 5 Stability of Starch Degrading Enzymes in Detergent

The ability of the starch degrading enzymes from strain 1E.1^(CT) towithstand detergents, which is essential for their application inlaundry detergents or textile desizing, is demonstrated.

100 ml Erlenmeyer flasks provided with a baffle were each charged with30 ml of 0.1M Na₂ CO₃ /NaHCO₃ buffer, pH 10.1 containing 0.12 g ofsodium dodecyl sulphate (equivalent to 4 g per liter). To one half ofthe flasks 0.3 g potato starch (equivalent to 1%) was added.

Each flask was dosed with enzyme-containing supernatant from strain1E.1^(CT) by adding 0.5, 1.0 or 2.0 ml (see Table 23). As a control, thesupernatant fluid was replaced with 1.0 ml water. Immediately afteradding the enzyme, a 0.1 ml sample was removed (time=zero hours) for themeasurement of enzyme activity.

The flasks were incubated with shaking at 25° C. for 2.5 hours at whichtime a second 0.1 ml sample was removed for the measurement of enzymeactivity.

As a comparison the experiment was repeated using a conventionalα-amylase derived from Bacillus subtilis.

Enzyme activity was determined using the reducing sugars methodpreviously described.

The results are recorded in Table 24.

                  TABLE 24                                                        ______________________________________                                        Stability of Starch Degrading Enzymes from Strain 1E.1.sup.CT in              Detergents                                                                    ENZYME-CONTAINING             ENZYME                                          SUPERNATENT                   UNITS RE-                                       ADDED           CONDI-        COVERED                                         (ml)            TIONS      pH     0 h. 2.5 h.                                 ______________________________________                                        0*                         10.4    0    0                                     0.5             SDS        10.3   26   20                                     1.0                        10.3   44   48                                     2.0                        10.3   109  113                                    0*                         10.3    0    0                                     0.5             SDS +      10.2   12   17                                     1.0             STARCH     10.1   36   48                                     2.0                        10.2   79   120                                    Standard § SDS    10.4    0      0                                       Standard § SDS + STARCH                                                                         10.2    0      0                                       ______________________________________                                         *replaced with 1 ml water                                                     § 2.8 RAU Bacillus subtilis α-amylase  One RAU (Reference          Amylase Unit) is defined as the quantity of enzyme that will convert 1 mg     of starch per minute at pH 6.6 and 30° C. into a product which upo     reaction with iodine has an equal absorbance at 620 nm. as a solution         containing 25 g CoCl.sub.2.6H.sub.2 O, 3.84 g K.sub.2 Cr.sub.2 O.sub.7 an     1 ml 1M HCl in 100 ml distilled water.                                   

The results of this test clearly demonstrate the stability of the starchdegrading enzymes, produced by the alkaliphilic bacterial strain of thepresent invention, in the presence of detergent.

EXAMPLE 6 Production of Lipolytic Enzymes

Eleven of the new strains which clearly exhibited lipase activity(Appendix D) were tested further for the production of lipolyticenzymes. The eleven strains are examples from Cluster 2 and Cluster 3(FIGS. 1A and 1B).

The experiments were carried out in 100 ml conical flasks containing 30ml sterile alkaline nutrient medium, pH 9.6, inoculated with theappropriate bacterial strain. Three different media were used,designated medium Z to BB (Appendix A). The flasks were placed in anorbital shaking incubator (300 rpm) at 30° C. for 48 hours.

The cells were separated from the culture broth by centrifugation andthe supernatant dialyzed against 50 volumes 0.1 mM Tris-HCl buffer pH 9,with 3 changes of buffer over 24 hours. The dialysate was freeze driedto give a lipase preparation (Table 25).

The lipase preparations obtained according to this example were used forthe washing test described in Example 7, below.

                  TABLE 25                                                        ______________________________________                                        Production of Lipase                                                                    PRODUCTION    LIPASE    LIPASE                                      STRAIN    MEDIUM        TLU/ml*   TLU/g                                       ______________________________________                                        39E.3     BB            1.3       134                                         40E.3     Z             1.2       118                                         41E.3     Z             1.1        82                                         42E.3     Z             1.2        76                                         44E.3     Z             1.2        99                                         45E.3.sup.CT                                                                            AA            1.4        98                                         48E.3     BB            2.0       152                                         49N.3     BB            1.5       123                                         50N.3     BB            2.0       100                                         51N.3     BB            1.0        98                                         52N.3     BB            1.2       128                                         ______________________________________                                         *TLU = True Lipase Unit as defined in U.S. Pat. No. 4,933,287.           

The results of this test clearly demonstrate the presence of lipolyticenzymes, produced by alkaliphilic bacteria of the present invention, inthe culture broth and in a freeze-dried preparation of the dialyzedculture broth.

EXAMPLE 7 Lipase Washing Test

The lipase preparations from Example 6 were tested for performance underwashing conditions in TIDE® powder (1.5 g/l), a detergent product fromProcter & Gamble.

The washing test (SLM-test) was carried out as described in U.S. Pat.No. 4,933,287, which is hereby incorporated by reference. As control, alipase derived from Pseudomonas alcaligenes strain M1 (CB3 473.85) asdescribed in U.S. Pat. No. 4,933,287 was used. The results are shown inTable 26.

                  TABLE 26                                                        ______________________________________                                        Lipase Washing Test                                                           Detergent: TIDE ® (powder), 1.5 g/l                                       Lipase: 2 TLU/ml                                                              Ca.sup.2+ : 10.sup.-5 M (sodium tripolyphosphate added)                       pH: 9.5                                                                                RECOVERY (%)                                                         STRAIN     TRIGLYCERIDES TOTAL LIPID                                          ______________________________________                                        39E.3      55.3          73.4                                                 40E.3      82.0          89.9                                                 41E.3      44.7          78.7                                                 42E.3      55.5          74.8                                                 44E.3      6.6           76.9                                                 45E.3.sup.CT                                                                             81.6          92.9                                                 48E.3      76.5          82.9                                                 49N.3      72.4          82.0                                                 50N.3      50.5          76.6                                                 51N.3      80.4          87.6                                                 52N.2      77.0          84.7                                                 M1         88.5          91.5                                                 control*   98.7          98.7                                                 ______________________________________                                         *Standard tap water as defined in U.S. Pat. No. 4,933,287                

The decrease in the percent recovery of triglycerides and total lipids,as compared to the control, clearly indicate the ability of thelipolytic enzymes, produced by the alkaliphilic bacteria of the presentinvention, to break down and remove triglycerides and their degradationproducts embedded on a fabric sample, as well as their improvedperformance as compared to a known lipase.

    ______________________________________                                        Appendix A                                                                    Media Used in the Present Invention                                           ______________________________________                                        MEDIUM A                                                                      Glucose                 10.0     gl.sup.-1                                    Peptone (Difco: Detroit, MI, USA)                                                                     5.0      gl.sup.-1                                    Yeast Extract (Difco)   5.0      gl.sup.-1                                    K.sub.2 HPO.sub.4       1.0      gl.sup.-1                                    MgSO.sub.4.7H.sub.2 O   0.2      gl.sup.-1                                    NaCl                    40.0     gl.sup.-1                                    Na.sub.2 CO.sub.3       10.0     gl.sup.-1                                    *Agar                   20.0     gl.sup.-1                                    MEDIUM B                                                                      Glucose                 10.0     gl.sup.-1                                    Peptone (Difco)         5.0      gl.sup.-1                                    Yeast Extract (Difco)   5.0      gl.sup.-1                                    K.sub.2 HPO.sub.4       1.0      gl.sup.-1                                    MgSO.sub.4.7H.sub.2 O   0.2      gl.sup.-1                                    NaCl                    40.0     gl.sup.-1                                    Na.sub.2 CO.sub.3       10.0     gl.sup.-1                                    Novobiocin              50.0     mgl.sup.-1                                   Agar                    20.0     gl.sup.-1                                    MEDIUM C                                                                      Glucose                 10.0     gl.sup.-1                                    Peptone (Difco)         5.0      gl.sup.-1                                    Yeast Extract (Difco)   5.0      gl.sup.-1                                    K.sub.2 HPO.sub.4       1.0      gl.sup.-1                                    MgSO.sub.4.7H.sub.2 O   0.2      gl.sup.-1                                    NaCl                    40.0     gl.sup.-1                                    Na.sub.2 CO.sub.3       10.0     gl.sup.-1                                    Lactalbumin             10.0     gl.sup.-1                                    Agar                    20.0     gl.sup.-1                                    MEDIUM D                                                                      Glucose                 10.0     gl.sup.-1                                    Peptone (Difco)         5.0      gl.sup.-1                                    Yeast Extract (Difco)   5.0      gl.sup.-1                                    K.sub.2 HPO.sub.4       1.0      gl.sup.-1                                    MgSO.sub.4.7H.sub.2 O   0.2      gl.sup.-1                                    NaCl                    40.0     gl.sup.-1                                    Na.sub.2 CO.sub.3       10.0     gl.sup.-1                                    Casein                  20.0     gl.sup.-1                                    Agar                    20.0     gl.sup.-1                                    MEDIUM E                                                                      Soluble Starch          10.0     gl.sup.-1                                    Peptone (Difco)         5.0      gl.sup.-1                                    Yeast Extract (Difco)   5.0      gl.sup.-1                                    K.sub.2 HPO.sub.4       1.0      gl.sup.-1                                    MgSO.sub.4.7H.sub.2 O   0.2      gl.sup.-1                                    NaCl                    40.0     gl.sup.-1                                    Na.sub.2 CO.sub.3       10.0     gl.sup.-1                                    Lactalbumin             10.0     gl.sup.-1                                    Agar                    20.0     gl.sup.-1                                    MEDIUM F                                                                      Soluble Starch          10.0     gl.sup.-1                                    Peptone (Difco)         5.0      gl.sup.-1                                    Yeast Extract (Difco)   5.0      gl.sup.-1                                    K.sub.2 HPO.sub.4       1.0      gl.sup.-1                                    MgSO.sub.4.7H.sub.2 O   0.2      gl.sup.-1                                    NaCl                    40.0     gl.sup.-1                                    Na.sub.2 CO.sub.        10.0     gl.sup.-1                                    Casein                  20.0     gl.sup.-1                                    Agar                    20.0     gl.sup.-1                                    MEDIUM G                                                                      Oxbile (Oxoid: Basingstoke, U.K.)                                                                     10.0     gl.sup.-1                                    (NH.sub.4).sub.2 SO.sub.4                                                                             5.0      gl.sup.-1                                    MgSO.sub.4.7H.sub.2 O   0.2      gl.sup.-1                                    Yeast Extract (Difco)   0.5      gl.sup.-1                                    Lactalbumin             10.0     gl.sup.-1                                    Agar                    20.0     gl.sup.-1                                    Adjusted to pH 8.5 with 50% Na.sub.2 CO.sub.3 solution                        MEDIUM H                                                                      Oxbile (Oxoid)          10.0     gl.sup.-1                                    (NH.sub.4).sub.2 SO.sub.4                                                                             5.0      gl.sup.-1                                    MgSO.sub.4.7H.sub.2 O   0.2      gl.sup.-1                                    Yeast Extract (Difco)   0.5      gl.sup.-1                                    Casein                  20.0     gl.sup.-1                                    Agar                    20.0     gl.sup.-1                                    Adjusted to pH 8.5 with 50% Na.sub.2 CO.sub.3 solution                        MEDIUM I                                                                      Sabouroud Dextrose Agar (Oxoid)                                                                       65.0     gl.sup.-1                                    NaCl                    40.0     gl.sup.-1                                    Na.sub.2 CO.sub.3       20.0     gl.sup.-1                                    Cycloheximide           50.0     mgl.sup.-1                                   Penicillin G            25000    IUl.sup.-1                                   Streptomycin            25       mgl.sup.-1                                   MEDIUM J                                                                      Bacto Potato Dextrose Agar (Difco)                                                                    39.0     gl.sup.-1                                    NaCl                    40.0     gl.sup.-1                                    Na.sub.2 CO.sub.3       20.0     gl.sup.-1                                    Novobiocin              50.0     mgl.sup.-1                                   MEDIUM K                                                                      Bacto Potato Dextrose Agar (Difco)                                                                    39.0     gl.sup.-1                                    NaCl                    40.0     gl.sup.-1                                    Na.sub.2 CO.sub.3       20.0     gl.sup.-1                                    Cycloheximide           50.0     mgl.sup.-1                                   Penicillin G            25000    IUl.sup.-1                                   Streptomycin            25.0     mgl.sup.-1                                   MEDIUM L                                                                      Glucose                 0.2.     gl.sup.-1                                    Peptone (Difco)         0.1      gl.sup.-1                                    Yeast Extract (Difco)   0.1      gl.sup.-1                                    K.sub.2 HPO.sub.4       1.0      gl.sup.-1                                    MgSO.sub.4.7H.sub.2 O   0.2      gl.sup.-1                                    NaCl                    40.0     gl.sup.-1                                    Na.sub.2 CO.sub.3       10.0     gl.sup.-1                                    Casein                  20.0     gl.sup.-1                                    Agar                    20.0     gl.sup.-1                                    MEDIUM M (pH 9.6)                                                             Oxbile (Oxoid)          2.0      gl.sup.-1                                    (NH.sub.4).sub.2 SO.sub.4                                                                             1.0      gl.sup.-1                                    MgSO.sub.4.7H.sub.2 O   0.04     gl.sup.-1                                    Yeast Extract (Difco)   0.1      gl.sup.-1                                    Olive Oil               10.0     ml l.sup.-1                                  Na.sub.2 CO.sub.3       6.1      gl.sup.-1                                    Agar                    20.0     gl.sup.-1                                    MEDIUM N (pH 9.6)                                                             Oxbile (Oxoid)          2.0      gl.sup.-1                                    (NH.sub.4).sub.2 SO.sub.4                                                                             1.0      gl.sup.-1                                    MgSO.sub.4.7H.sub.2 O   0.04     gl.sup.-1                                    Yeast Extract (Difco)   0.1      gl.sup.-1                                    Olive Oil               10.0     ml l.sup.-1                                  Na.sub.2 CO.sub.3       6.1      gl.sup.-1                                    Tergitol 7 (Fluka: Buchs, CH)                                                                         500      ppm                                          Agar                    20.0     gl.sup.-1                                    MEDIUM O (pH 9.6)                                                             Oxbile (Oxoid)          2.0      gl.sup.-1                                    (NH.sub.4).sub.2 SO.sub.4                                                                             1.0      gl.sup.-1                                    MgSO.sub.4.7H.sub.2 O   0.04     gl.sup.-1                                    Yeast Extract (Difco)   0.1      gl.sup.-1                                    Olive Oil               10.0     ml l.sup.-1                                  Na.sub.2 CO.sub.3       6.1      gl.sup.-1                                    NaCl                    40.0     gl.sup.-1                                    Agar                    20.0     gl.sup.-1                                    MEDIUM P (pH 9.6)                                                             oxbile (oxoid)          10.0     gl.sup.-1                                    (NH.sub.4).sub.2 SO.sub.4                                                                             5.0      gl.sup.-1                                    MgSO.sub.4.7H.sub.2 O   0.2      gl.sup.-1                                    Yeast Extract (Difco)   0.5      gl.sup.-1                                    Olive Oil               10.0     ml l.sup.-1                                  NaCl                    40.0     gl.sup.-1                                    Agar                    20.0     gl.sup.-1                                    Adjusted to pH 9.6 with 50% Na.sub.2 CO.sub.3 solution                        MEDIUM Q (pH 9.6)                                                             Oxbile (Oxoid)          10.0     gl.sup.-1                                    (NH.sub.4).sub.2 SO.sub.4                                                                             5.0      gl.sup.-1                                    MgSO.sub.4.7H.sub.2 O   0.2      gl.sup.-1                                    Yeast Extract (Difco)   0.5      gl.sup.-1                                    Olive Oil               10.0     ml l.sup.-1                                  Agar                    20.0     gl.sup.-1                                    Adjusted to pH 9.6 with 50% Na.sub.2 CO.sub.3 solution                        MEDIUM R (pH 9.5)                                                             Fresh Yeast             82.5     gl.sup.-1                                    Glucose                 3.3      gl.sup.-1                                    K.sub.2 HPO.sub.4       1.6      gl.sup.-1                                    K.sub.2 CO.sub.3        0.6      gl.sup.-1                                    KHCO.sub.3              1.76     gl.sup.-1                                    CaCl.sub.2              0.05     gl.sup.-1                                    MgSO.sub.4.7H.sub.2 O   0.05     gl.sup.-1                                    FeSO.sub.4              0.005    gl.sup.-1                                    MnSO.sub.4              0.0066   gl.sup.-1                                    MEDIUM S                                                                      Fresh Yeast             8.25     gl.sup.-1                                    Glucose                 1.32     gl.sup.-1                                    K.sub.2 HPO.sub.4       1.6      gl.sup.-1                                    CaCl.sub.2              0.05     gl.sup.-1                                    MgSO.sub.4.7H.sub.2 O   0.05     gl.sup.-1                                    FeSO.sub.4              0.005    gl.sup.-1                                    MnSO.sub.4              0.0066   gl.sup.-1                                    NaCl                    40.0     gl.sup.-1                                    Adjusted to pH 10.5 with 40% Na.sub.2 CO.sub.3                                solution                                                                      MEDIUM T (pH 10.1)                                                            Glucose                 10.0     gl.sup.-1                                    Peptone (Difco)         5.0      gl.sup.-1                                    Yeast Extract (Difco)   5.0      gl.sup.-1                                    K.sub.2 HPO.sub.4       1.0      gl.sup.-1                                    MgSO.sub.4.7H.sub.2 O   0.2      gl.sup.-1                                    NaCl                    40.0     gl.sup.-1                                    Na.sub.2 CO.sub.3       10.0     gl.sup.-1                                    MEDIUM U                                                                      Oxbile                  10.0     gl.sup.-1                                    (NH.sub.4).sub.2 SO.sub.4                                                                             5.0      gl.sup.-1                                    MgSO.sub.4.7H.sub.2 O   0.2      gl.sup.-1                                    Yeast extract (Difco)   0.5      gl.sup.-1                                    Casein                  10.0     gl.sup.-1                                    Adjusted to pH 9.8 with 40% Na.sub.2 CO.sub.3 solution                        MEDIUM V                                                                      Tryptone Soya Broth (Oxoid)                                                                           30.0     gl.sup.-1                                    Adjusted to pH 9.9 with 40% Na.sub.2 CO.sub.3 solution                        MEDIUM W (pH 10.1)                                                            Soluble starch          10.0     gl.sup.-1                                    Peptone (Difco)         5.0      gl.sup.-1                                    Yeast extract (Difco)   5.0      gl.sup.-1                                    KH.sub.2 PO.sub.4       1.0      gl.sup.-1                                    MgSO.sub.4.7H.sub.2 O   0.2      gl.sup.-1                                    NaCl                    40.0     gl.sup.-1                                    Na.sub.2 CO.sub.3       10.0     gl.sup.-1                                    MEDIUM X                                                                      Skim milk (Difco)       100.0    gl.sup.-1                                    Adjusted to pH 10.8 with 25% Na.sub.2 CO.sub.3                                solution                                                                      MEDIUM Y                                                                      Yeast Extract (Difco)   1.0      g                                            KNO.sub.3               10.0     g                                            KH.sub.2 PO.sub.4       1.0      g                                            MgSO.sub.4.7H.sub.2 O   0.2      g                                            Na.sub.2 CO.sub.3       10.0     g                                            NaCl                    40.0     g                                            Soluble starch (Merck)  20.0     g                                            Demineralised water     1        liter                                        MEDIUM Z                                                                      Brain Heart Infusion (Difco)                                                                          20.0     g                                            Na.sub.2 EDTA (Komplexion III, Siegfried AG,                                  Switzerland)            1.0      g                                            FeSO.sub.4.7H.sub.2 O   0.006    g                                            MnSO.sub.4.H.sub.2 O    0.003    g                                            CaCl.sub.2.2H.sub.2 O   1.0      g                                            MgSO.sub.4.7H.sub.2 O   0.25     g                                            Tween 80                5.0      g                                            Soya oil                5.0      g                                            Distilled water to 1 liter, pH of medium                                      adjusted to pH 9.6 with 25% Na.sub.2 CO.sub.3                                 solution.                                                                     MEDIUM AA                                                                     Yeast Extract (Difco)   20.0     g                                            KH.sub.2 PO.sub.4       5.0      g                                            FeSO.sub.4.7H.sub.2 O   0.006    g                                            MnSO.sub.4.H.sub.2 O    0.003    g                                            CaCl.sub.2.2H.sub.2 O   1.0      g                                            MgSO.sub.4.7H.sub.2 O   0.25     g                                            Tween 80                5.0      g                                            Soya oil                5.0      g                                            Distilled water to 1 liter, pH of medium                                      adjusted to pH 9.6 with 25% Na.sub.2 CO.sub.3                                 solution.                                                                     MEDIUM BB                                                                     Brain Heart Infusion (Difco)                                                                          20.0     g                                            FeSO.sub.4.7H.sub.2 O   0.006    g                                            MnSO.sub.4.H.sub.2 O    0.003    g                                            CaCl.sub.2.2H.sub.2 O   1.0      g                                            MgSO.sub.4.7H.sub.2 O   0.25     g                                            Tween 80                5.0      g                                            Soya oil                5.0      g                                            Distilled water to 1 liter, pH of medium                                      adjusted to pH 9.6 with 25% Na.sub.2 CO.sub.3                                 solution.                                                                     ______________________________________                                         *(when required for a solid medium)                                      

APPENDIX B Methods for Unit Tests

1. Character numbers 1 to 5

Colony color, form, elevation, margin, size

A suspension of bacteria was spread over an alkaline nutrient agar(Medium A) and cultivated at 37° C. Colonies were examined after 48hours.

2. Character number 6 and 7

Cell morphology, Gram's strain reaction

Bacteria cells grown in alkaline nutrient broth (Medium A, without agar)for 24 hours were spun down in a centrifuge and resuspended in a smallamount of alkaline nutrient broth and allowed to air-dry on a microscopeslide. Or, bacteria were cultivated for 24-48 hours on an alkalinenutrient agar (Medium A) so as to form colonies. Colonies of bacteriawere suspended in physiological saline and a few drops allowed toair-dry on a microscope slide. The Gram's staining test was performedusing the Dussault modification (Journal of Bacteriology, 70, 484-485,1955) with safranin as counterstain.

3. Character number 8

Oxidase reaction

Filter paper moistened with a 1% aqueous solution ofN,N,N',N'-tetramethyl-p-phenylenediamine or oxidase identification discs(bioM erieux: Charboni eres-les-Bains, France) were smeared with a youngbacterial culture from alkaline nutrient agar. A purple color within 1minute was recorded as a positive reaction. E. coli, used as a control,did not give a positive reaction within one minute.

4. Character number 9

Skim milk test

A minimal medium composed (g/l distilled water) of yeast extract, 1.0;KNO₃, 10.0; K₂ HPO₄, 1.0; MgSO₄.7H₂ O, 0.2; NaCl, 40.0; Na₂ CO₃, 10.0;agar, 20.0 was supplemented with 5.0 g/l skim milk powder, sterilised byautoclaving and poured into Petri dishes. Bacteria were inoculated andincubated at 37° C. Areas of clearing around bacterial colonies in anotherwise opaque agar were recorded as a positive reaction.Non-alkaliphilic reference strains were tested in an identical fashionusing media of the same composition but without Na₂ CO₃ so as to give apH of 6.8-7.0.

5. Character number 10

Gelatin hydrolysis

Charcoal-gelatin discs (bioM erieux) or "chargels" (Oxoid) wereincubated at 37° C. in an alkaline nutrient broth (Medium A) togetherwith bacteria. A black sediment indicated a positive reaction.

6. Character number 11

NaCl tolerance

Two methods were applied.

(a) Bacterial strains were cultivated at 37° C. on an alkaline nutrientagar (Medium A) containing 0%, 4%, 8%, 12% or 15% (w/v) NaCl. The agarplates were examined for bacterial growth after 48 hours.

(b) Bacterial strains were cultivated at 37° C. in an alkaline nutrientbroth (Medium A) containing 0%, 4%, 8%, 12%, 15% or 25% NaCl. Bacterialgrowth was monitored by optical density measurements using a Klett meter(green filter) at 0, 12, 24, 48, 72 and 144 hours.

7. Character number 12

Minimum pH for growth

Nutrient agar, pH 6.8-7.0 (Medium A without sodium carbonate) was pouredinto square Petri dishes. A strip of solidified agar was removed fromone end and replaced with molten 4% (w/v) agar containing 3.6% (w/v) Na₂CO₃ and 0.8% (w/v) NaOH. A pH gradient from pH 10.5 to pH 7 across theplate was allowed to develop overnight. Bacteria were inoculated bystreaking along the pH gradient and cultivated at 37° C. for 48 hours.The pH at the point where bacterial growth ceased was measured with aflat head electrode and with "Alkalite" pH strips (Merck: Darmstadt, W.Germany).

8. Character numbers 13-21

Carbohydrate utilisation

A minimal medium composed (g/l distilled water) of yeast extract, 1.0;KNO₃, 10.0; K₂ HPO₄, 1.0; MgSO₄.7H₂ O, 0.2; NaCl, 40.0; Na₂ CO₃, 10.0;agar, 20.0 was supplemented with 2.0 g/l of the carbohydrate under testand poured into square Petri dishes. Bacteria were inoculated, using a25 point multipoint inoculator, from 1.0 ml of a bacterial suspensioncultivated for 48 hours in an alkaline nutrient broth (Medium A). Theagar-plates were incubated at 37° C. for 48 hours. The results wererecording by comparing bacterial growth on minimal nutrient mediumcontaining a carbohydrate supplement with growth on a minimal mediumwithout the carbohydrate under test.

Non-alkaliphilic reference strains were tested in an identical fashionusing media of the same composition but without Na₂ CO₃ so as to give apH of 6.8-7.0.

9. Character numbers 22-53

Growth on carbon substrates

Use was made of the commercially available test strip ATB 32 GN(API-bioM erieux: La Balme les Grottes, France). The strips were usedaccording to the manufacturer's instructions but with an addition of 1.0ml of a solution containing 4% NaCl and 1% Na₂ CO₃ to the vials of basalmedium provided. The strips were incubated at 37° C. for 48 hours.Non-alkaliphilic reference strains were incubated in the standard basalmedium.

10. Character numbers 54-72

Enzymatic activities

Use was made of the commercially available test strip APIZYM (API-bioMerieux) which was used according to the manufacturer's instructions,except that the alkaliphilic bacterial cells were suspended in alkalinenutrient broth (Medium A). The strips were incubated at 37° C. for 4hours.

11. Character numbers 73-82

Amino acids as carbon and nitrogen source

The same technique was employed as for tests 14-21 except that KNO₃ wasomitted from the minimal nutrient medium.

12. Character numbers 83-104

Antibiotic sensitivity

A light suspension of bacteria in alkaline nutrient broth was spread onthe surface of alkaline nutrient agar (Medium A) and allowed to dry.Commercially available antibiotic susceptibility test discs (Oxoid orMast Laboratories: Merseyside, U.K.) were applied to the agar surface.The bacteria were cultivated at 37° C. for 48 hours. Clear zones aroundthe antibiotic disks indicated sensitivity.

    ______________________________________                                        Appendix C                                                                    Unit Tests for Analysis by Numerical Taxonomy                                 CHAR-                                                                         ACTER   TEST               COMPUTER                                           NUMBER  DESCRIPTION        CODE                                               ______________________________________                                         1      Colony color       white = 1                                                                     cream = 2                                                                     beige = 3                                                                     yellow = 4                                                                    orange = 5                                                                    pink = 6                                                                      brown = 7                                                                     red = 8                                             2      Colony form        circular = 1                                                                  irregular = 2                                                                 punctiform = 3                                                                filamentous = 4                                     3      Colony elevation   convex = 1                                                                    raised = 2                                                                    umbonate = 3                                                                  flat = 4                                            4      Colony margin      entire = 1                                                                    undulate = 2                                                                  lobate = 3                                                                    fimbriate = 4                                       5      Colony size        diameter in                                                                   millimeters                                         6      Cell morphology    rod = 1                                                                       coccus = 2                                          7      Gram's stain       negative = 1                                                                  positive = 2                                        8      Oxidase test       negative = 1                                                                  positive = 2                                        9      Skim milk test     negative = 1                                                                  positive = 2                                       10      Gelatin hydrolysis negative = 1                                                                  positive = 2                                       11      NaCl tolerance     growth at                                                                     0%-4% = 1                                                                     growth at                                                                     0%-8% = 2                                                                     growth at                                                                     0%-12% = 3                                                                    growth at                                                                     0%-15% = 4                                                                    growth only at                                                                0% = 5                                                                        growth only at                                                                4%-15% = 6                                         12      Minimum pH for     pH 7.5 = 7.5                                               growth on nutrient agar                                                                          pH 8.0 = 8.0                                                                  pH 8.5 = 8.5                                                                  pH 9.0 = 9.0                                                                  pH 9.5 = 9.5                                                                  pH 10.0 = 10.0                                                                pH 10.5 = 10.5                                     13-21   Carbohydrate utilisation                                              13      Formate                                                               14      Fumarate                                                              15      Succinate                                                             16      Galactose          enhanced                                                                      growth = 2                                         17      Pyruvate           equal growth = 1                                   18      Fructose           growth                                                                        inhibited = 0                                      19      Lactose                                                               20      Xylose                                                                21      Starch                                                                22-53   Growth on carbon substrates                                           22      Rhamnose                                                              23      N-acetylglucosamine                                                   24      D-ribose                                                              25      Inositol                                                              26      D-saccharose                                                          27      Maltose                                                               28      Itaconate                                                             29      Suberate                                                              30      Malonate                                                              31      Acetate                                                               32      DL-lactate         positive = 2                                       33      L-alanine          negative = 1                                       34      Mannitol                                                              35      D-glucose                                                             36      Salicin                                                               37      D-melibiose                                                           38      L-fucose                                                              39      D-sorbitol                                                            40      L-arabinose                                                           41      Propionate                                                            42      Caprate                                                               43      Valerate                                                              44      Citrate                                                               45      Histidine                                                             46      5-ketogluconate                                                       47      Glycogen                                                              48      3-hydroxybenzoate                                                     49      L-serine                                                              50      2-ketogluconate                                                       51      3-hydroxybutyrate                                                     52      4-hydroxybenzoate                                                     53      L-proline                                                             54-72   Enzymatic activities                                                  54      Alkaline phosphatase                                                  55      Esterase (C4)                                                         56      Esterase lipase (C8)                                                  57      Lipase (C14)                                                          58      Leucine arylamidase                                                   59      Valine arylamidase                                                    60      Cystine arylamidase                                                                              positive = 2                                       61      Trypsin            negative = 1                                       62      Chymotrypsin                                                          63      Acid phosphatase                                                      64      Naphthol-AS-BI-                                                               phosphohydrolase                                                      65      α-galactosidase                                                 66      β-galactosidase                                                  67      β-glucuronidase                                                  68      α-glucosidase                                                   69      β-glucosidase                                                    70      N-acetyl-β-glucosaminidase                                       71      α-mannosidase                                                   72      α-fucosidase                                                    73-82   Amino acids as carbon and                                                     nitrogen source                                                       73      Serine                                                                74      Proline                                                               75      Asparagine                                                            76      Arginine           enhanced                                                                      growth = 2                                         77      Alanine            equal growth = 1                                   78      Lysine             no growth = 0                                      79      Methionine                                                            80      Phenylalanine                                                         81      Glycine                                                               82      Valine                                                                 83-104 Antibiotic sensitivity                                                83      Gentamycin    10     μg                                            84      Nitrofurantoin                                                                              50     μg                                            85      Ampicillin    25     μg                                            86      Nalidixic Acid                                                                              30     μg                                            87      Sulphamethoxazole                                                                           50     μg                                            88      Trimethoprim  2.5    μg                                            89      Penicillin G  1      IU  antibiotic sensitive                         90      Chloramphenicol                                                                             25     μg                                                                             inhibition of                                                                 growth = 2                                   91      Erythromycin  5      μg                                            92      Fusidic Acid  10     μg                                                                             antibiotic                                                                    insensitive,                                 93      Methicillin   10     μg                                                                             no                                                                            growth                                                                        inhibition = 1                               94      Novobiocin    5      μg                                            95      Streptomycin  10     μg                                            96      Tetracycline  25     μg                                            97      Sulphafurazole                                                                              100    μg                                            98      Oleandomycin  5      μg                                            99      Polymixin     300    IU                                               100     Rifampicin    2      μg                                            101     Neomycin      30     μg                                            102     Vancomycin    30     μg                                            103     Kanamycin     30     μg                                            104     Bacitracin    10     IU                                               ______________________________________                                    

    ______________________________________                                        Appendix D                                                                    Screening for Proteolytic, Amylolytic and Lipolytic Activity                  ______________________________________                                        Proteolytic Activity                                                          STRAIN LACTALBUMIN   CASEIN   BLOOD  GELATIN                                  ______________________________________                                        Cluster 1                                                                     1E.1.sup.CT                                                                          +             +        +      +                                        2E.1   -             -        -      -                                        wB2    -             -        -      +                                        wB5    -             -        -      -                                        wBs4   -             +        +      +                                        10B.1  +             +        +      +                                        20N.1  +             +        -      +                                        27M.1  -             +        -      -                                        wNk2   -             +        -      +                                        Cluster 2                                                                     39E.3  n.t.          n.t.     n.t.   +                                        41E.3  n.t.          n.t.     n.t.   +                                        45E.3.sup.CT                                                                         n.t.          n.t.     n.t.   +                                        47E.3  n.t.          n.t.     n.t.   +                                        51N.3  n.t.          n.t.     n.t.   +                                        52N.3  n.t.          n.t.     n.t.   +                                        42E.3  n.t.          n.t.     n.t.   +                                        50N.3  n.t.          n.t.     n.t.   +                                        Cluster 3                                                                     6B.1   +             +        -      +                                        7B.1   +             +        -      -                                        8B.1   -             +        -      +                                        38E.2  n.t.          n.t.     n.t.   -                                        56E.4  +             n.t.     n.t.   -                                        25B.1  n.t.          +        n.t.   +                                        26N.1  -             +        -      +                                        11C.1  +             +        -      +                                        wB.1   -             -        -      +                                        12C.1  -             +        -      -                                        28N.1.sup.CT                                                                         -             -        -      -                                        61N.4  +             n.t.     n.t.   -                                        36E.2  n.t.          n.t.     n.t.   -                                        40E.3  n.t.          n.t.     n.t.   +                                        65B.4  +             n.t.     n.t.   -                                        94LM.4 n.t.          n.t.     n.t.   +                                        19N.1  -             +        -      +                                        24B.1  +             +        -      +                                        21M.1  +             +        +      +                                        Cluster 3                                                                     29C.1  -             -        -      -                                        35E.2  n.t.          n.t.     n.t.   -                                        37E.2  n.t.          n.t.     n.t.   -                                        48E.3  n.t.          n.t.     n.t.   +                                        78LN.4 n.t.          +        n.t.   +                                        73aC.4 n.t.          +        n.t.   +                                        75C.4  n.t.          +        n.t.   +                                        73bC.4 n.t.          +        n.t.   +                                        74C.4  n.t.          +        n.t.   -                                        77LN.4 n.t.          +        n.t.   -                                        wN1    -             -        -      -                                        49N.3  n.t.          n.t.     n.t.   +                                        44E.3  n.t.          n.t.     n.t.   +                                        58E.4  n.t.          n.t.     n.t.                                            57E.4  +             n.t.     n.t.   +                                        Cluster 4                                                                     wE5    -             -        -      +                                        wB4.sup.CT                                                                           -             -        -      -                                        wNk1   -             -        -      +                                        wE11   -             -        -      -                                        wE12   -             -        -      +                                        Cluster 5                                                                     9B.1   -             +        -      +                                        16N.1  +             n.t.     n.t.   +                                        17N.1.sup.CT                                                                         +             +        -      +                                        22M.1  +             +        -      +                                        Cluster 6                                                                     18N.1  +             +        -      +                                        59E.4  +             n.t.     n.t.   +                                        64B.4.sup.CT                                                                         +             n.t.     n.t.   +                                        63N.4  +             n.t.     n.t.   +                                        53E.4  +             n.t.     n.t.   +                                        Non-Clustering Strains                                                        wN.2   -             -        -      +                                        4E.1   -             -        -      -                                        5E.1   -             -        -      +                                        92LM.4 n.t.          n.t.     n.t.   +                                        wBn5   -             -        -      +                                        ______________________________________                                        Amylolytic and Lipolytic Activity*                                                              LIPASE                                                             STARCH     ACTIVITY   ESTERASE LIPASE                                         HYDROL-    ON         LIPASE   ACTI-                                   STRAIN YSIS       OLIVE OIL  ACTIVITY VITY                                    ______________________________________                                        Cluster 1                                                                     1E.1.sup.CT                                                                          +          -          +        +                                       2E.1   +          -          +        -                                       wB2    -          -          +        -                                       wB5    -          -          +        -                                       wBs4   -          -          +        -                                       10B.1  +          -          +        -                                       20N.1  -          -          +        -                                       27M.1  -          -          +        -                                       wNk2   +          -          +        -                                       Cluster 2                                                                     39E.3  +          +          +        -                                       41E.3  +          +          +        -                                       45E.3.sup.CT                                                                         +          +          +        +                                       47E.3  +          +          -        -                                       51E.3  +          +          +        +                                       52E.3  +          +          +        +                                       42E.3  +          +          +        +                                       50N.3  +          +          +        +                                       Cluster 3                                                                     6B.1   -          -          +        -                                       7B.1   +          -          +        +                                       8B.1   +          +          +        +                                       38E.2  -          -          +        -                                       Cluster 3                                                                     56E.4  -          +          +        +                                       25B.1  -          -          +        +                                       26N.1  -          -          +        -                                       11C.1  -          -          +        -                                       wB1    -          -          +        -                                       12C.1  +          -          -        -                                       28N.1.sup.CT                                                                         +          -          +        -                                       61N.4  +          -          -        -                                       36E.2  -          -          -        -                                       40E.3  +          +          +        +                                       65B.4  +          -          +        -                                       94LM.4 +          -          +        -                                       19N.1  +          -          +        +                                       24B.1  -          -          +        -                                       21M.1  +          -          -        -                                       29C.1  +          -          +        +                                       35E.2  -          -          +        -                                       37E.2  -          -          +        -                                       48E.3  -          +          +        -                                       78LN.4 -          -          +        -                                       73aC.4 -          -          +        --                                      75C.4  +          -          -        -                                       73bC.4 +          -          +        -                                       74C.4  +          -          +        -                                       77LN.4 -          -          +        +                                       wN1    +          -          +        -                                       49N.3  +          +          +        -                                       44E.3  +          +          +        +                                       58E.4  +          +          +        -                                       57E.4  +          -          +        -                                       Cluster 4                                                                     wE5    +          -          -        -                                       wB4.sup.CT                                                                           -          -          +        -                                       wNk1   +          -          +        -                                       wE11   -          -          +        -                                       wE12   -          -          +        -                                       Cluster 5                                                                     9B.1   +          -          +        -                                       16N.1  +          -          +        -                                       17N.1.sup.CT                                                                         +          -          +        -                                       22M.1  +          -          +        -                                       Cluster 6                                                                     18N.1  +          -          +        -                                       59E.4  +          -          +        -                                       64B.4.sup.CT                                                                         +          -          +        -                                       63N.4  +          -          -        -                                       53E.4  +          -          +        -                                       Non-Clustering Strains                                                        wN2    +          -          +        +                                       4E.1   -          -          +        -                                       5E.1   +          -          +        -                                       92LM.4 +          +          +        -                                       wBn5   -          -          +        -                                       ______________________________________                                         n.t. = not tested                                                             *Starch Hydrolysis determined according to Character 21 (Appendix B)          Lipase Activity (Olive Oil) determined on media M-P (Appendix A)              Esterase Lipase Activity determined according to Character 56 (Appendix B     Lipase Activity determined according to Character 57 (Appendix B)        

    ______________________________________                                        Appendix E                                                                    Percentage Positive States for Characters in Clusters                                      CLUSTER                                                          CHARACTER      1      2      3    4    5    6                                 ______________________________________                                        [6]  Cell morphology                                                                             9      0    12   0    0    0                               [7]  Gram's stain  0      0    0    0    0    0                               [8]  Oxidase test  55     100  35   80   0    20                              [9]  Skim milk test                                                                              36     11   6    20   0    20                              [10] Gelatin       67     100  56   60   100  100                             [13] Formate       0      0    15   40   0    0                               [14] Fumarate      45     78   74   80   75   60                              [15] Succinate     45     89   85   40   67   50                              [16] Galactose     45     11   12   40   25   0                               [17] Pyruvate      64     89   88   40   75   0                               [18] Fructose      45     11   68   60   50   100                             [19] Lactose       9      0    0    0    0    0                               [20] Xylose        18     11   15   20   0    0                               [21] Starch        73     100  91   20   100  100                             [22] Rhamnose      9      0    15   20   0    0                               [23] N-            9      0    26   20   0    100                                  acetylglucosamine                                                        [24] D-ribose      27     0    50   0    0    20                              [25] Inositol      9      0    9    0    50   0                               [26] D-saccharose  27     0    74   20   25   100                             [27] Maltose       9      11   74   40   50   100                             [28] Itaconate     0      11   6    0    0    0                               [29] Suberate      9      67   53   40   0    0                               [30] Malonate      0      11   65   0    25   40                              [31] Acetate       18     100  100  80   25   100                             [32] DL-lactate    27     56   100  100  0    40                              [33] L-alanine     18     89   82   100  0    100                             [34] Mannitol      0      0    41   20   0    80                              [35] D-glucose     9      11   71   60   0    60                              [36] Salicin       0      0    3    20   0    60                              [37] D-melibiose   0      0    3    0    0    0                               [38] L-fucose      0      0    0    20   0    20                              [39] D-sorbitol    18     0    41   0    0    40                              [40] L-arabinose   9      0    3    40   0    0                               [41] Propionate    9      100  94   80   0    80                              [42] Caprate       0      78   32   40   0    80                              [43] Valerate      9      100  97   80   0    40                              [44] Citrate       9      56   94   20   50   100                             [45] Histidine     0      0    71   0    0    80                              [46] 5-ketogluconate                                                                             0      0    21   0    0    0                               [47] Glycogen      9      22   26   20   25   100                             [48] 3-hydroxybenzoate                                                                           9      0    38   0    0    0                               [49] L-serine      0      0    68   60   0    0                               [50] 2-ketogluconate                                                                             0      0    56   80   25   20                              [51] 3-hydroxybutyrate                                                                           18     33   94   100  0    100                             [52] 4-hydroxybenzoate                                                                           0      0    71   80   0    0                               [53] L-proline     45     100  100  80   25   80                              [54] Alkaline      100    44   94   40   100  60                                   phosphatase                                                              [55] Esterase (C4) 100    100  100  100  100  100                             [56] Esterase lipase (C8)                                                                        100    89   85   100  100  100                             [57] Lipase (C14)  9      67   26   0    0    0                               [58] Leucine arylamidase                                                                         91     67   94   60   50   0                               [59] Valine arylamidase                                                                          91     33   65   100  25   0                               [60] Cystine arylamidase                                                                         73     0    15   0    0    0                               [61] Trypsin       64     11   9    60   0    0                               [62] Chymotrypsin  73     0    3    20   75   0                               [63] Acid phosphatase                                                                            91     11   94   100  100  60                              [64] Naphthol      91     22   3    20   100  40                                   phosphohydrolase                                                         [65] α-galactosidase                                                                       0      11   3    20   100  0                               [66] B-galactosidase                                                                             9      0    0    20   100  0                               [67] β-glucuronidase                                                                        9      0    3    20   25   0                               [68] α-glucosidase                                                                         27     0    79   60   100  40                              [69] β-glucosidase                                                                          9      0    9    80   75   0                               [70] N-acetyl-β-                                                                            27     0    0    0    0    0                                    glucosaminidase                                                          [71] α-mannosidase                                                                         0      0    0    0    0    0                               [72] α-fucosidase                                                                          9      0    0    20   0    0                               [73] Serine        22     13   29   60   100  50                              [74] Proline       56     63   65   80   100  100                             [75] Asparagine    67     38   61   60   75   100                             [76] Arginine      56     25   53   80   50   50                              [77] Alanine       67     100  62   40   100  50                              [78] Lysine        44     75   71   80   75   100                             [79] Methionine    60     50   53   nc   50   50                              [80] Phenylalanine 89     100  76   100  100  100                             [81] Glycine       44     13   29   80   50   50                              [82] Valine        44     50   41   0    50   25                              [83] Gentamycin    36     67   3    20   0    0                               [84] Nitrofurantoin                                                                              18     33   21   0    0    0                               [85] Ampicillin    45     67   56   0    100  80                              [86] Nalidixic Acid                                                                              36     78   76   0    0    20                              [87] Sulphamethoxazole                                                                           18     33   38   0    0    0                               [88] Trimethoprim  45     22   35   0    75   60                              [89] Penicillin G  27     11   29   0    75   100                             [90] Chloramphenicol                                                                             100    100  100  100  100  100                             [91] Erythromycin  91     100  100  100  100  100                             [92] Fusidic Acid  18     0    3    20   100  100                             [93] Methicillin   45     11   50   0    100  100                             [94] Novobiocin    18     0    3    0    25   0                               [95] Streptomycin  100    78   97   100  75   100                             [96] Tetracycline  45     22   3    20   100  100                             [97] Sulphafurazole                                                                              40     13   20   nc   0    0                               [98] Oleandomycin  100    38   94   100  100  100                             [99] Polymixin     89     63   78   100  0    0                               [100]                                                                              Rifampicin    100    38   78   100  100  100                             [101]                                                                              Neomycin      0      13   0    0    0    0                               [102]                                                                              Vancomycin    22     13   0    20   100  75                              [103]                                                                              Kanamycin     11     13   0    0    0    0                               [104]                                                                              Bacitracin    33     13   0    20   100  100                             ______________________________________                                    

We claim:
 1. A pure culture consisting of a single strain of bacteriauseful for the production of alkali-tolerant enzymes wherein the strainis aerobic, Gram-negative, and rod-shaped, and is an obligatealkaliphile that has the following characteristics:a) formscream-colored, circular colonies; b) grows optimally between pH9 andpH10; c) gives a positive response to the following tests:1) leucinearylamidase 2) Valine arylamidase 3) Phosphohydrolase 4) Polymixin; d)gives a negative response to the following tests:1) N-acetylglucosamine2) Maltose 3) Propionate 4) Caprate 5) Valerate 6) Citrate 7) Histidine8) Glycogen 9) 4-hydroxybenzoate 10) α-galactosidase.
 2. A single strainof bacteria which is useful for the production of alkali-tolerantenzymes, wherein said strain is aerobic, Gram-negative, and rod-shaped,and is an obligate alkaliphile that has the following characteristics:a)forms cream-colored, circular colonies; b) grows optimally between pH9and pH10; c) gives a positive response to the following tests:1) leucinearylamidase 2) Valine arylamidase 3) Phosphohydrolase 4) Polymixin; d)gives a negative response to the following tests:1) N-acetylglucosamine2) Maltose 3) Propionate 4) Caprate 5) Valerate 6) Citrate 7) Histidine8) Glycogen 9) 4-hydroxybenzoate 10) α-galactosidase.
 3. A pure cultureconsisting of a single strain of bacteria useful for the production ofalkali-tolerant enzymes, wherein the strain is aerobic, Gram-negative,and rod-shaped, is an obligate alkaliphile, forms cream-colored circularcolonies, grows optimally between pH9 and pH10 and is classified at a73% similarity level into a phenon based on a positive response to thefollowing tests:1) leucine arylamidase 2) Valine arylamidase 3)Phosphohydrolase 4) Polymixin;and on a negative response in thefollowing tests: 1) N-acetylglucosamine 2) Maltose 3) Propionate 4)Caprate 5) Valerate 6) Citrate 7) Histidine 8) Glycogen 9)4-hydroxybenzoate 10) α-galactosidase.
 4. A pure culture consisting of asingle strain of bacteria useful for the production of alkali-tolerantenzymes, wherein the strain is aerobic, Gram-negative, and rod-shaped,is an obligate alkaliphile, forms cream-colored circular colonies, growsoptimally between pH9 and pH10 and is classified at a 73% similaritylevel into a phenon based on the pattern of responses to tests leaving aprobability matrix with a percentage distribution as follows:1)N-acetylglucosamine--13 2) Saccharose--25 3) Maltose--25 4) Lactate--385) Propionate--0 6) Valerate--13 7) Citrate--13 8) Histidine--0 9)Glycogen--0 10) 3-hydroxybutyrate--13 11) 4-hydroxybenzoate--0 12)Leucine arylamidase--88 13) Valine arylamidase--88 14)Phosphohydrolase--88 15) α-galactosidase--0 16) Ampicillin--50 17)Fusidic Acid--25 18) Methicillin--50 19) Polymixin--88 20)Vancomycin--13 21) Yellow colony--0 22) Translucent colony--0 23)Lipase--0 24) Oxidase (10 secs)--25.